© 2016 Nature America, Inc. All rights reserved. suggest suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy. J.H. J.H. ( University Medical Center Hamburg, Germany.Hamburg-Eppendorf, Experimental Hematology, Hannover Medical School, Hannover, Germany. 6 Hamburg, Germany.Hamburg-Eppendorf, Hamburg, Germany. 1 most HIV-1 infected individuals. concept, Tre recombinase is not suitable to eradicate the provirus from application of Tre recombinase. Hence, although providing proof of an from the benefit potentially could patientsHIV-1of infected 1% than sequence is not conserved amongvery different HIV-1 clades and less in the LTR ( togenerate nase Tre recombinase that recombines present sequence a recombiCreofDNA the bindingredirecting specificity feasibilityof causing undesired side Our previouseffects. work had established the majority of HIV-1 isolates and efficiently removes the provirus without described havebeen tools ing egies to excise the integrated provirus using advanced genome engineerapproaches to control or novel preferablydeveloping in cureinterest angrowing HIV a infection. is cART,Several there of strat limitations activation, along with associated long-term adverse effects immune of levels increased exhibit still cART on patients of number treatmentadherence the emergence of drug resistance is discontinued medication when observed is rebound viral provirus, integrated the as resting memory CD4 replication-competent viruses persists in long-lived HIV reservoirs,viduals such infection using cART, present therapies do not cure HIV-infected indi Although substantial advances have been made in the treatment of HIV efficacious on clinical HIV-1 isolates HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant from infected cells, we employed 145 cycles of directed substrate-linked evolution to evolve a recombinase (Brec1) that site- as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus Current combination therapies antiretroviral (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists Axel Schambach Karl Hackmann Deboyoti Chakraborty Janet Karpinski provirus of most H Directed evolution of a recombinase that excises the nature biotechnology nature Received 30 April 2015; accepted 21 December 2015; published online 22 February 2016; Structural Bioinformatics, Structural BIOTEC Bioinformatics, TU Dresden, Germany. Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. [email protected] 1– 3 . A small population of latent, transcriptionally silent but still loxLTR 6 . The challenges of lifelong cART include drug toxicity 3 German German Center for Infection Research (DZIF), partner site Hamburg, Germany. ) of a primary HIV-1 isolate 1 5 11 , 8 , , Evelin Schrock 2 , , Christoph Lindner , , 1 11 + 2 T cells and high costs andhigh , , Ilona Hauber

1 advance online publication online advance , Niklas , Beschorner Niklas 15– 4 , 5 1 8 . As current treatment does not target 8 , 9 . However, the target. these ofnone , adverse drug interactions 5 ) ) or F.B. ( Institute Institute for Clinical Genetics, University Hospital and Medical Faculty Carl Gustav Carus, TU Dresden, Germany. 1 3 in in vitro 5 . Furthermore, . substantial a , , Josephine Abi-Ghanem 2 I [email protected] , 11 V-1 primary isolates with high specificity 1 5 9 , , Jan Chemnitz . However, the , , Jan van Lunzen and 7 Max Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany. in in vivo 2 , , Helga Hofmann-Sieber 1 4 11 . Given the These These authors contributed equally to this work. should Correspondence be addressed to , , including in mice humanized with cells. patient-derived Our data 1

0 loxLTR , strict 9 Agaplesion Diakonieklinikum, Gynecology,Agaplesion Diakonieklinikum, Hamburg, Germany. 2 , 11 7 - - - - , 2

, , , Carola Schäfer ). 3 , 10 than most other sequences within the HIV-1 genome. the within sequences other most than exact the to be highly conserved and thus less susceptible match to mutational changes isolates A ( subtype sequence of 198) of out (158 subtype B viruses, 85% (745 out of 876) of subtype C isolates, and 80% to homology sequence ( ( systems to similarity remote its and C, and B A, groups subtype HIV-1 region major target Brec the called we ( which sequence, Sequence a HIV selected We Alamos Los the ( in Databases libraries long sequence HIV-1 in screened algorithm SeLOX sequence the employed we target (LTRs) repeats conserved terminal highly a identify To selection Target site RESULTS therapies. future in application its favor particularly that and, as a byrecombinase, is Cre-type properties characterized several evolution molecular by directed evolved was Brec1 isolates. HIV-1 primary of majority the against active is that (Brec1) 1 recombinase 6 i. 1 Fig. loxBTR , , M Teresa Pisabarro , , Joachim Hauber The The We now report the development and application of the broad-range loxP 2 Heinrich Heinrich Pette Institute, Leibniz Institute for Experimental Virology, d loxBTR b and target sequences of related site-specific recombinase recombinase site-specific related of sequences target and oi:10.1038/nbt.346 ) due to its high conservation rate (90%) throughout the the throughout (90%) rate conservation high its to due ) ) and is a 34-bp asymmetric sequence displaying 32% 32% displaying sequence asymmetric 34-bp a is and ) Fig. 1 Fig. Fig. 1 Fig. http://www.hiv.lanl.gov 4 2 Department Department of Anesthesiology, University Medical Center , sequence is located in the R region of HIV-1 LTRs HIV-1 of region R the in located is sequence 3 a , , Ulrike C Lange c ) 2 and 15 , 3 , 20– , , Maciej Paszkowski-Rogacz Supplementary Table 1 Supplementary 2 loxP 2 2 . , 7 3 & Frank Buchholz 6 . As 94% (1,297 out of 1,386 isolates) of of isolates) 1,386 of out (1,297 94% As . , , Vineeth Surendranath 2 / – ) for appropriate candidates. candidates. appropriate for ) 4 , , Adam Grundhoff 10 ), this sequence seems seems sequence ), this Infectious Infectious Diseases Unit, 8 Institute Institute of 1 ticles e l c i rt A , 7 1 , 7 ,

loxBTR

1 2 9 and and , 3 , 

© 2016 Nature America, Inc. All rights reserved. clones for their recombination properties in in properties recombination their for clones specificities. novel with enzymes produce to efforts future ate recombinases evolve to target different and sequences should acceler mutational hot spots substantially enhances our understanding of how recombinase Tre previous Cre evolution studies face in the Cre/ specificity site target in ( changes for critical likely as regions four Cre/ the onto mutations the ( N317, P307, D278, T268, E262, N319, I320) being altered with a mutation R259, frequency of 85% or higher A249, K244, R241, N160, A175, C155, S108, G93, K86, Y77, S51, M44, H40, M30, P15, P12, V7, (N3, residues 26 with process, evolution the during fixed been in the protein. Furthermore, the results unmasked mutations that had regions different of evolvability divergent the highlight results these ( process evolution the ize of Brec sequences were obtained and aligned to Cre in order to visual on forming deep sequencing the library.final A total of reads 331,000 mutations by per conserved evolutionarily and identified resolution properties. desired the with mutants obtain to required were cycles ( tolerated well is expression long-term that ensure to weeks 4 for recombinase the expressing constitutively ( sites target related of recombination against counter-selection with off-target activity, evolutionpotential cycling was continued reduce forTo 18 additional generations cycles. evolution 126 after obtained final the on ( recombinases probe ultimately to collapsed progressively were sites the and shuffled, and pooled were Fig. 1b by PCR and retrieved ligated back into the initial pEVOselectively plasmids ( were sequences coding recombinase DNA, active plasmid isolated the of digestion NdeI After site. restriction tive sites recombined the plasmid and thereby removed a unique NdeI and sites tor 1A-Ctarget pEVO and the starting 2A-C containing ( evolution experiments all available recombinase coding sequences from previous Cre-related ess ( which were successively collapsed during the directed evolution proc process evolution city previously been addressed in studies to alter Cre DNA binding specifi recognition site The recombinases HIV-1 broad-range of evolution Directed A, B, C (blue discs), and of ( site target Brec1 the of LTRs location with the DNA and gray.in ( shown are sequences to compared sites, half enzyme-bound ( ( 1 Figure s e l c i rt A  (Brec1, clone One c loxP a Fig. Fig. 2 Fig. Fig. 2 Fig. 2 Fig. Fig. 2 Fig.

) Representations (%) of the target sequences shown in ) Brec1 target site ( site target ) Brec1 From the final library (cycle 145) we tested more than 100 individual To investigate frequently appearing alterations in detail, we mapped nucleotide at process evolution molecular the investigated We on activity good possessing library recombinase A 15 Fig. 2 Supplementary Fig. 1 ) and related recombinase target sites. Sequence differences in the the in differences Sequence sites. target recombinase related ) and loxBTR , 20– a c b a ). After accumulation of functional recombinases the libraries the libraries recombinases of ). functional accumulation After ). ). These regions are located at the protein-DNA binding inter

). ). Finally, the library was transduced into human Jurkat T cells and and and and 2 Highly conserved recombinase target site site target recombinase conserved Highly 3 a . This divergence necessitated initiating the substrate-linked and site differs in 23 out of the 34 nucleotides of the Cre Cre the of nucleotides 34 the of out 23 in differs site Supplementary Fig. 1 Fig. Supplementary Supplementary Table 2 Supplementary loxP Supplementary Fig. 1 loxP 1 20 5 ( system, and they compared evolved differently to loxBTR , and 11 alterations in the inverted repeats had not 2 Fig. 2b Fig. 4 15 with six different subsites (1A-C and 2A-C), 2A-C), and (1A-C subsites different six with upeetr Fg 2 Fig. Supplementary , 20 loxBTR , ). Recombinases with activity on the respec ) sequence aligned to the Cre target site site target Cre the to aligned ) sequence 2 Fig. 2 Fig. 5 b loxP and inserted them into the evolution vec , c ) Schematic representation of proviral proviral of representation ) Schematic 20 ). Indeed, identification of these four four these of identification Indeed, ). in the three individual main subtypes. , 21 crystal structure crystal b , ). The high resolution afforded by afforded resolution high The ). 2 5 ). loxP , including the previously evolved ). As a starting library we pooled ). Fig. 2 Fig. , are marked in red. The spacer spacer The red. in marked , are a ). Thus, 145 evolution evolution 145 Thus, ). loxBTR ) was selected for for selected was ) 26 loxBTR a shrci coli. Escherichia , loxBTR in HIV-1 subtypes 2 Supplementary Supplementary 7 in HIV-1 in LTRs. . We identified We. identified loxBTR sequence sequence .

Fig. Fig. 2 was was a ------

subsequent experiments based on its promising recombination recombination the promising of recombination its efficient showing on behavior, based experiments subsequent enhanced green fluorescent protein (EGFP) was expressed only only expressed was by recombination. sequence (EGFP) encoding of upon the mCherry excision protein fluorescent green enhanced whereas plasmid, reporter nonrecombined the from expressed tively constitu was (mCherry) protein fluorescent red monomeric assay, vated cell sorting (FACS) ( ( by microscopy fection reporter plasmids. Recombinase activity was evaluated 48 h after trans cells, HeLa mammalian cells were co-transfected with in recombinase expression and Brec1 of properties recombination the examine To recombines specifically Brec1 Brec1. of proficiency recombination do not isolates, the compromise found in primary those in particular its to similar was on variants activity these on Brec1 of activity the Notably, ( the in mutations loxBTR occurring recurrently four on selected we Brec1 of mutants, behavior recombination the assess To Brec1. by recognition escape might sequence target this in mutations with recombines specifically and efficiently Brec1 that demonstrating systems, nase recombi related of sites target on cross-reactivity showing without recombined proficiently also Brec1 results, above the with ( recombination signal to assay reporter white into mammalian cells. Third, we performed experiments using a blue/ vectors lentiviral (SIN) inactivating carrying loxBTR ( sites target different on ( level induction at highest the observed recombination full with increased, gressively in levels induction different upon of Brec1 on We its site target efficiency the recombination tested first targets specifically Brec1 recombining without Fig. 3 Fig. c b a Despite the high sequence conservation of of conservation sequence high the Despite Host DNA lo lo ro lo lo lo lo xBTR xL xM7 xH xP x lo xM7 d sites, whereas no recombination was detected in plasmids plasmids in detected was recombination no whereas sites, sequence from the Los Alamos HIV Sequence Database Database Sequence HIV Alamos Los the from sequence TR xP advance online publication online advance ) and tested the ability of Brec1 to recombine these sites. sites. these recombine to Brec1 of ability the tested and ) loxP 0% 5 loxBTR Br lo ′ ro

loxBTR LT xH ec1 x , , R loxLTR 1 Fig. 3 Fig. ( Halfsit in bacteria. in Fig. 3 Fig. LT , or SIN or , R loxP a Fig. 4 Fig. e ). Second, we assessed Brec1 recombination recombination Brec1 assessed we Second, ). U3 HIV Fig. 3 Fig. e Fig. Fig. 4 . ), suggesting that certain point mutations, point mutations, that certain ), suggesting Subt -1 pr lo <1% loxBTR xL 1 a ∆ 80% 3 TR ) and quantified by fluorescence-acti ) quantified and ype lox, a a lox, o b b viral genom ). ). In the fluorescence-based reporter ). Brec1 efficiently recombined the the recombined efficiently Brec1 ). Spacer loxBTR A E. coli E. 28 lo and variant sites in bacteria in sites variant and R loxBTR xBTR , 2 9

designed for Brec1 delivery delivery Brec1 for designed 2 e . Brec1 recombination pro recombination Brec1 . U5 2 Subt nature biotechnology nature in mammalian cells mammalian in 94% -like site present in self- in present site -like ype Halfsit loxBTR lo B Fig. 3 Fig. 90% xBTR e loxBTR 3 Br ′ , some viruses viruses some , c LT Subt 3 ec1 ). Consistent Consistent ). 4 R 85% ype sequence sequence Br Tr Dr Cr C2#4 Fr e loxBTR loxBTR e ec1 e e C - - - - -

© 2016 Nature America, Inc. All rights reserved. only in cell cultures that were transduced with lentiviral or or lentiviral with or (LV-Brec1 transduced vectors (LV- vectors control RV-Brec1), negative with but treated not in cells were GFP-expressing and that Brec1- cultures alpharetroviral cell in observed were only products recombination circular excised Precisely genomes that HIV-1 cells full-length integrated reporter stably HeLa contained in activity Brec1 analyzed we Moreover, in ( HeLa cells constructs reporter integrated of stably recombination we tested cells, of eukaryotic genome the in ( systems recombinase loxBTR in As letters. the of size the by represented frequencies acid amino with shown in as yellow, and blue green, magenta, in shown are residues mutated Frequently enlarged. shown regions hotspot Tre during evolution in listed are mutations acid amino conserved All conservation. acid amino specific no with but three, position acid amino in mutations of occurrence high marks triangle The acids. amino possible different two with but site a specific in mutation in used colors the with correlating magenta, or blue green, yellow, in shown hotspots mutational with black colored are process evolution directed the during (>85%) mutations conserved evolutionarily of Bars library. Brec the in (%) occurrence of frequency ( evolution. of rounds loxBTR through recombination B, gels. the beside indicated are bands triangle) one with (line recombined the and triangles) two with (line nonrecombined The arrow. the below presented is subset each on performed cycles evolution of number The gels. agarose respective of images as shown are sites 2 Figure nature biotechnology nature RV-Ctr; or Ctr b a

To investigate whether Brec1 can also recombine Mutation frequency 100% 100% 25% 50% 75% 85% 85% 75% 50% 25% 0% E. coli E. ; H, recombination through through recombination ; H, target sites, with no cross-reactivity on target sites of related related of sites target on cross-reactivity no with sites, target

2 2 Brec1 evolution. ( evolution. Brec1 1C 1B 1A loxBTR 2C 2B 2A Complex libraryofrandomly cycles 1–42 in evolutionvectorpEVO , Brec1 efficiently and site-specifically recombined its its recombined site-specifically and efficiently Brec1 , mutated recombinases 4 0 L L -ara -ara Fig. 4d Fig. subsites 7 7 6 0 1 5 b is provided. ( provided. is ) Deep sequencing of the final Brec library. Each bar represents an amino acid mutation compared to the Cre sequence and its its and sequence Cre the to compared mutation acid amino an represents bar Each library. Brec final the of sequencing ) Deep 8 0 Combine Fig. 4a Fig. , shuffle e and ). 0 a ) Temporal scheme of the evolution process. The recombinase library activities of the first and last cycle of the target target the of cycle last and first the of activities library recombinase The process. evolution the of ) Temporal scheme

100 advance online publication online advance loxBTR 1b 1a cycles 43–78 , 2b 2a b 120 ). loxH L L c -ara -ara ) Structural analysis of mutational hotspots. The structure of Cre bound to to bound Cre of structure The hotspots. mutational of analysis ) Structural subsites 140 9 9 ; P, recombination through through ; P, recombination 160 Residue * Combine shuffle 180 and Supplementary Fig. 3 Supplementary 200 loxBTR cycles 79–109 2 1 loxBTR 220 L L -ara -ara subsites 14 17 240 * sites present 2 9 Combine ( 260 shuffle and Fig. 4 Fig.

loxP 280 Sequence analysisofevolvedlibrary Identification ofmutationalhotspots . Triangles labeled . labeled Triangles c Concentrated 300 Brec library ). ). ). ). B loxP Cycles 110–126

active Asymmetric 320

target site L

loxBTR base positions 1 and 13 as shown in in shown as 13 1 and positions base T cells by lentiviral Jurkat vector in gene transfer Brec1 (LV-cBrec1) expressed for up constitutively to we 10 weeks First, effects. or genotoxic cytopathic cytotoxic, conceivable assess to experiments formed per we approach, Brec1 the of effects side potential Toinvestigate evaluations Safety infection. cells. cultured in HIV-1 activity anti HIV-1 possesses and cells malian on Brec1 of recombines specifically and efficiently Brec1 effect Thus, antiviral the documenting release was abrogated specifically in Brec1-expressing cells ( cells Brec1-expressing in particle specifically abrogated was release Viral virus. replication-competent with infected research, HIV-1 in used routinely line T-cell human a cultures, T-lymphocyte -ara

Brec library Brec Tre library Tre 17 340 We next tested whether Brec1 can inhibit HIV-1 in human PM1 PM1 human in HIV-1 inhibit can Brec1 whether tested Wenext Cycle 145 B B c Brec Cre Tre Efficient, highlyspecificBreclibrarycompatible l 240 257 -ara indicate reduced levels of of levels reduced indicate -ara with highexpressioninhumancells A249 K244 Supplementary Table 2 Supplementary counter selection cycles 127–144 R241 B/H B/P Specificity 264 249 L L -ara -ara 9 9 c . Asterisks mark high occurrence of a of occurrence high mark . Asterisks R259 loxP G263 E262 P B H B Functional analysisofindividualclones A260 b 1 . Alignments to Cre and Tre and Cre to are . Alignments is depicted with the four mutational mutational four the with depicted is Figure 1 Figure . A comparison to the mutations mutations the to . A comparison l Selection ofBrec1 13 -arabinose for progressive progressive for -arabinose a Tolerance validation are labeled. are (lentiviral delivery) in humanTcells H4 0 M4 4 ticles e l c i rt A K4 loxBTR 3 G9 84 beam Laser Q8 3 9 39 K86 + A84 in mam in Fig. 4 Fig. Detector – 45 95 f ), ),  - -

© 2016 Nature America, Inc. All rights reserved. T cells, derived from an HIV-1 patient, that had been transduced transduced been had that patient, HIV-1 an from derived cells, T CD4 primary on reads billion 1.3 over totaling sequencing genome proviruses are harboring several rare. Moreover, we whole performed 6 Fig. distant in one ofNo cell. of recombination two viruses integration reflecting multiple between bination recom potential to investigate analyses we performed Brec1- Furthermore, in ( alterations cells chromosomal expressing no found and (array-CGH) hybridization genomic array-comparative and (SKY) karyotyping CD4 transduced ous 5b Fig. in Table3 Supplementary to similarity sequence highest the SeLOX algorithm for genome human the We screened effects. lineages ( expected the into differentiate to HSC human of capacity colony using the not did alter Brec1 that lineages demonstrated assays (CFU) unit forming hematopoietic various into differentiate to CD34 Brec1-vector–transduced of capacity the analyzing Furthermore, cell. host the in tolerated well is Brec1 of expression prolonged that indicating experiments, these of any in observed were cells transduced control and Brec1 between ( function and activation immune and apoptosis expression, gene to respect with CD4 cycle ( cell progression and growth cell on impact potential Brec1’s assayed and triangles. two or one with a line as indicated are bands 1 mg/ml with induced Brec1, of activity to aligned are Sequences sequence. each below indicated ( expression. lacZ driving promoter the of removal to due colonies white in results Recombination plates. X-gal on grown were panels) the beside indicated (as plasmids reporter site target and expression recombinase with co-transformed in assay reporter recombination l 1 mg/ml at grown were sites target indicated and sequence Brec1-encoding the containing 4). (lane cells mammalian in delivery Brec1 for designed vector expression a lentiviral in present site a mutated and 3) 2, (lanes sites target recombinase related control), positive 1, (lane ( panel. lower on Treof recombinase recombination comparison, For triangles). (two plasmids nonrecombined for fragment larger a and triangle) (one recombined for fragment a smaller in resulting digestion, enzyme restriction by assayed was DNA plasmid isolated of Recombination 1–4). (lanes concentrations indicated at grown were sites target and sequence Brec1-encoding the ( vector pEVO on Brec1 of ( bacteria. in specific 3 Figure s e l c i rt A  Supplementary Fig. 4h Fig. Supplementary -arabinose. M, DNA marker. ( marker. DNA M, -arabinose.

Second, Second, we analyzed potential Brec1-related genotoxic or off-target . coli E. loxBTR + T lymphocytes were transduced with Brec1 and analyzed analyzed and Brec1 with transduced were lymphocytes T ), indicating that Brec1-mediated genomic deletions in cells cells in deletions genomic Brec1-mediated that indicating ), ), indicating that the human genome does not contain obvi contain not does genome human the that indicating ),

loxBTR E. coli E. Brec1 recombination is efficient and and efficient is recombination Brec1 Nn o tee ie wr rcmie ( recombined were sites these of None . loxBTR -like sequences that are targeted by Brec1. We tested by Brec1. also targeted are that sequences -like b Supplementary Fig. 1b Fig. Supplementary Supplementary Fig. 4a Fig. Supplementary ) Activity of Brec1 on on Brec1 of ) Activity cells harboring the pEVO vector vector pEVO the harboring cells Supplementary Fig. 4c Fig. Supplementary sites was detected in this scenario ( scenario this in detected was sites + T cells for genomic potential alterations by spectral . . upeetr Fg 5c Fig. Supplementary E. coli E. a ) Recombination efficacy efficacy ) Recombination loxLTR 1 9 . . Six human genomic sites (HGS 1–6) with the ) were tested for recombination proficiency proficiency recombination for tested were ) ). cells harboring the the harboring cells is shown in the the in shown is l c -arabinose -arabinose loxBTR ) LacZ-based ) LacZ-based E. coli E. loxBTR ) containing ) containing loxBTR + . Cells . Cells sites integrated in the genome, genome, the in integrated sites hematopoietic stem cells (HSC) (HSC) cells stem hematopoietic loxBTR l d , loxBTR -arabinose, on on -arabinose, b ( – ) Frequent ) Frequent ). Likewise, primary human human primary Likewise, ). Supplementary Fig. 5a Fig. Supplementary f

). No significant difference difference significant No ).

and data not shown). shown). not data and

loxBTR loxBTR loxBTR Viral variants: a d b loxBTR VS4 VS3 VS2 VS1 Supplementary Supplementary Supplementary Supplementary -like sites using using sites -like loxBTR Brec1 nucleotide alterations found in different clinical HIV-1 isolates, with subtype frequency frequency subtype with HIV-1 isolates, clinical different in found alterations nucleotide Tre -like sites VS1, VS2, VS3 and VS4 (lanes 2–5). The nonrecombined and recombined recombined and nonrecombined The 2–5). (lanes VS4 and VS3 VS2, VS1, sites -like kb Halfsite kb . Nucleotides that differ from from differ that . Nucleotides 4 5 4 5 4 5 1 M 1 M and and µ g/ml 1 0 + - -

loxBTR

L -arabinose Spacer ‘personalized’ immunodeficient mice by transplanting Rag2 transplanting by mice immunodeficient ‘personalized’ HIV-1 isolates demonstrate that Brec1 can ( efficiently excise proviral cells DNA of primary Brec1-treated the in depleted efficiently was DNA proviral the that revealed indeed period culture 20-d the after cells the from DNA isolated genomic of sequencing Deep cells. showed LV-Brec1 with ( transduced cells decreased viral loads and same stable amounts of transduced cells over time the contrast, In cells. LV-Ctr–transduced of viability cells and growth the ( transduced compromised of time number over the and decreased increased loads viral cells, In LV-Ctr–transduced Methods). Online 20 see d (for details, patient LV-Ctr LV-Brec1or ( eral of blood an HIV-1–infected patient and the transduced cells with ject. To mirror this situation we isolated CD4 clinical setting Brec1 would have human to load function in viral an monitor already infected subsubsequently to vector-transduced strains research, therapy hematoly gene antiviral In cells patient-derived in ablation provirus Brec1-mediated effects. genotoxic or cytotoxic cytopathic, apparent sion is well tolerated in human cells and in a model organism without tolerated in an organism. Together, the data that signify Brec1 expres is well that expression Brec1 of more 18 than demonstrating months, ited at a Mendelian ratio with animals remaining healthy over a period health of the animals at frequent The intervals. Brec1 allele was inher ( promoter constitutive ( alterations genetic mediated Brec1- inadvertent of signs no LV-Ctr,found LV-Brec1 or and with 0 3 2 3 2 Fig. Fig. 5 loxP To test efficacy of this approach in an an in approach this of efficacy test To a from Brec1 express that mice transgenic generated we Finally, 100

loxLTR b 1,000 advance online publication online advance ), ), indicating Brec1-mediated provirus excision in the patient’s 4 m 4 Halfsite SIN phoid cells are frequently infected with established HIV-1phoid with cells are established infected frequently ∆ lox in vitro.in loxBTR c Fig. 5 Fig. Brec1 are shown in red. ( red. in shown are Cre Tr Supplementary Fig. 7 Fig. Supplementary i. 5 Fig. e e a ) and subsequently cultured the cells for cells the cultured subsequently and ) Supplementary Table 4 Supplementary loxBTR b ), implying that viral expansion expansion viral that implying ), kb 4 5 Blue 1 M lacZ

e loxBTR ) Agarose gel showing the the showing gel ) Agarose nature biotechnology nature Recombination 16– 3 2 + VS1 loxLTR T cells from the periph 18 ) and monitored the the monitored and ) , Fig. 5 Fig. n vivo in 29– VS2 3 5 4 2 VS3 . However, in a in However, . c ). ). These data data These ). model, we we model, VS4 loxP White −/− lacZ γ c −/− - - - -

© 2016 Nature America, Inc. All rights reserved. n vco-rndcd (CD4 vector-transduced and (CD45 human of enrichment an revealed cells human of sis transplantation after ( 18–21 detection week of at limit copies/ml) the RNA HIV-1 below (<20 loads to time viral over plasma declined cells, continuously patient-derived Brec1-transduced with cells ( vector–transduced transplanted control were which into animal the of plasma the in viremia robust revealed animals individual the from obtained Viral CD45 human and loads transplantation. after weeks 4–8 (PBMC) cells mononuclear blood peripheral of Human FACSanalysis by LV-Brec1. verified was or LV-Ctrengraftment cell either with transduced and patient CD4 with (hu-Rag2) animals DNA. proviral HIV of depletion Brec1-mediated indicating chart), bar panel, (lower PCR real-time quantitative HIV-1 of amount the and cultures both from prepared was DNA genomic infection, 8 after week 1 and week At units. light relative RLU, 9 weeks. over panel) (middle supernatant the in antigen p24 of amount the as well as panel) (upper activity luciferase quantifying by monitored was Infection frame. reading open a luciferase the where HIV-1 CCR5-tropic the of ng 500 with 1 × 10 transduction, after 13 day At = 10). (MOI RV-Ctr RV-Brec1 or with transduced were Cells T lymphocytes. PM1 human ( in described as analyzed and RV-Brec1 RV-Ctr or with transduced were Cells delivery. Brec1 alpharetroviral upon recombination ( panel). lower (boxed; precise in resulted and LTRby a single of flanked presence the demonstrating sequenced, subsequently was LTR The region control. PCR negative NC, control; PCR positive PC, panel). upper kb; (1 product excision circular transient the detect to P2 and P1 primers using PCR by transduction after days indicated LV-Ctr with the at LV-Brec1or analyzed was transduced cells HeLa HIV-1 infected from ( site. integration the LTRat a single remains recombination, Upon product. excision circular transient the of detection permit P2) and (P1 primers PCR Provirus-specific recombination. ( experiments. independent three of mean bars, Error counts. cell to red counts cell of green ratio as the measured efficiency in view. of ( field same the from always are EGFP.of shown Images expression drive to promoter the allowing sequence, encoding mCherry the of excision to leads (triangles) sites target the at Recombination fluorescence. red only in resulting silent, is EGFP whereas expressed constitutively is mCherry recombination, to Prior shown. are plasmids reporter fluorescent and expression recombinase indicated with co-transfected cells HeLa of assay. Images reporter ( cells. 4 Figure nature biotechnology nature been had cells Brec1-expressing that indicating liver, and spleen the c f Fig. 5 Fig. ) Analysis of antiviral Brec1 activity in activity Brec1 antiviral of ) Analysis ) Schematic depiction of Brec1-mediated Brec1-mediated of depiction ) Schematic a were analyzed by FACS and recombination by FACS analyzed were recombination and gag a 6 d -leader sequences. Brec1 activity activity Brec1 sequences. -leader ) Fluorescence-based recombination recombination ) Fluorescence-based cells of each culture were infected infected were culture each of cells

). When the Brec1 animal was euthanized at week 21, analy 21, at week euthanized was animal Brec1 the When ). Brec1 recombination in mammalian mammalian in recombination Brec1 nef coding region is substituted by substituted is region coding d ) Genomic DNA isolated isolated DNA ) Genomic loxBTR i. 5 Fig. e ) Analysis of BTR BTR of ) Analysis + recombination recombination CD4 d

b ) Cells as shown shown as ) Cells ). By contrast, in the mouse engrafted engrafted mouse the in contrast, By ). advance online publication online advance + + + cells were monitored over time. Data Data time. over monitored were cells T cell pools derived from an HIV-1 an from derived pools cell T GFP

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sequences normalized to human hemoglobin subunit gamma ( gamma subunit hemoglobin human to normalized sequences

Brec e d c a Cr Tr 1 kb 1 kb 1 e e nef nef d0 d0 LTR U3 P1 LTR U3 220 Integrated provirus 220

+ d1 d1 loxBTR CD4 mCherry Red d2 d2 RV-Ctr LV-Ctr P2 d3 d3 + - ) PolyA site

d4 d4 720 d5 d5 transduced human immunodeficient/ CD34 combined described ously previ as transplants given and diabetic/severe irradiated were mice NSG) gamma; (nonobese SzJ NOD.Cg-antiviral newborn Brec1-based effects, CD34 of modification genetic include also may therapies gene Anti-HIV cell–transplants stem with animals in activity Brec1 patients. HIV-1–positive from isolated cells in provirus the target can Brec1 that show ments ( specific animal showed similar progression, validating that this effect is not patient or donors HIV-1–positive from two additional derived ments cells with experi transplantation Mouse organs. these in time over enriched loxBTR Brec1 720 loxBTR d6 d6 EGFP + R R HSC d0 d0 740 Genomic 740 d1 d1 loxLTR LTR Recombinatio 33 d2 d2 LV-Brec1 RV-Brec1 , 3 4 d3 d3 2 760 . . Therefore, in another 9 Supplementary Fig. 8 Fig. Supplementary 760 by intrahepatic injection of LV-Ctr– of LV-Brec1– or injection intrahepatic by d4 d4 Excision produc LTR U5 d5 LTR U5 d5 n 84 P2 d6 d6 0 + 840 HSC. In these experiments the transduction LT ga PC ga R PC P1 hbg g g loxP t leader leader 860 860 NC ) sequences was assessed by assessed was ) sequences NC Green EGFP 400 µ in vivo m ). Collectively, these experi these Collectively, ). f Brec1 b

approach to investigate 6 Cre ng p24/1 × 10 cells Tre

gag/hbg 1,000 5

0.01 RLU/1 × 10 cells 0.05 0.10 0.15 Prkdc 100 10 10 10 10 10 10 0. 10 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80 1 1 0 0 0 0 2 3 4 5 6 7 ticles e l c i rt A Weeks postinfectio Weeks postinfectio loxBTR Weeks postinfectio 0 0 loxBTR loxBTR 1 1 Recombination scid efficiency (%) RV-Brec RV-Ctr 2 2 1 RV-Brec RV-Ctr 7 6 5 4 3 3 IL2rg loxLTR loxLTR loxLTR 4 6 5 1 8 1 tm1Wjl loxP loxP loxP 9 8 7 8 n 9 n n  - - - /

© 2016 Nature America, Inc. All rights reserved. antiviral activity of Brec1 at the organism level. organism at the Brec1 of activity antiviral ( HSC LV-Brec1–transduced received had that mice demonstrated that p24 representative of spleens in cells antigen–expressing p24 HIV-1 and ( DNA proviral HIV of depletion Brec-1–mediated indicating reduced in the Brec1-transduced cells as quantified by real-time PCR, tion of HIV-1 ( DNA. As before genomic prepare to spleen the CD4 human and infection HIV-1 after Brec1- either vector, at or HSC were with week control euthanized 24 transduced vector engrafted animals additional mice, humanized engraftment. cell human the of loss to due not was animals Brec1-treated the in observed loads virus in decline the that demonstrating tissues, in various cells of transduced analysis again revealed a strong accumulation FACS euthanized, were animals these After ( load time over viral declined prominently the HSC, LV-Ctr–transduced as opposed to the control animal that received of p24 antigen) CCR5-tropic HIV-1 replication-competent of the injection were intraperitoneal blood by peripheral challenged their in phocytes CD45 human transplantation >10% with animals and after weeks 16 at analysis Human cell engraftment GFP was verified ~20% by FACS in resulted typically rate chart). (bar suspensions cell single- FACSby using necropsy at determined was animal Brec1-expressing the of liver and spleen PBMC, in cells human of Percentage time. over monitored were squares) (blue cells CD45 human of percentage and dots) CD4 patient-derived human (LV-Ctr vector LV-Brec1)-transduced or 1 × 10 of injection i.p. by transplants Rag2 ( regions. nonoverlapping three of is samples treated Error shown. bars, mean Brec1- and nontreated for counts average of Quantification indicated. ratios and counts the with cells, Brec1-treated and nontreated for lines as shown are (HXB2) genome HIV-1 the of and (control) 12 chromosome on regions 500-bp from reads sequencing panel in shown cultures the from transduction after 20 day at isolated DNA ( points. time indicated the at determined were squares) CD4 human transduced of percentage and dots) (red LV-Brec1.load Viral 1 × 10 with transduced and PBMC patient–derived × 10 (3 T cells ( arrows. by highlighted steps important with workflow, the of representation ( T cells. primary patient-derived 5 Figure s e l c i rt A  Fig. 6 Fig.

Altogether, the combined combined the Altogether, in effects Brec1-mediated antiviral demonstrate Todirectly more −/− 7 JR-CSF b γ infectious particles of LV-Ctrof or particles infectious

c ). Likewise, immunohistochemical analysis of human CD3 human of analysis immunohistochemical Likewise, ). Brec1 deletes the HIV-1 provirus from from HIV-1 provirus the deletes Brec1 −/− (hu-Rag2) mice were given given were mice (hu-Rag2) strain to equivalent (dose 200 ng c ) Deep sequencing of genomic genomic of sequencing ) Deep 6 gag ) were isolated from HIV-1 from isolated ) were 3 5 . For LV-Brec1–treated mice, sequences normalized to normalized sequences human + T cells. Viral load (red (red load Viral T cells. + GFP b + ) Primary CD4 ) Primary cells were depleted in distinctly animals + in vivo in cells (blue (blue cells a b d ) Schematic ) Schematic . Individual . Individual ) BALB/c ) BALB/c + 6 CD4 lentiviral lentiviral i. 6 Fig. experiments reveal substantial substantial reveal experiments

+

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Fig. 6 Fig. Fig. 4 Fig. a d b

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2 5 1 4 2 5 PBMC arising in an infected individual. Moreover, within the experimental experimental the Moreover, within individual. infected an in arising of development the resistance, because Brec1 about would still recognize at concerns least some mutationslower also might feature This mutations ( target. Brec1 a be would individuals affected million 28 than more ( 82% of representation overall an with subtypes HIV-1 other most in served contain the exact the into fall C or B A, subtypes M worldwide HIV-1group individuals infected of 72% Over isolates. site ( target conserved a and highly recombining recognizing by cells specifically HIV-1–infected from provirus the remove can Brec1 that We show including HIV, may be the only way replication-competent a reservoirs, to latent cure. genuine viral achieve of elimination Complete DISCUSSION The fact that Brec1 can also recognize recognize also can Brec1 that fact The 0 8 0 s 0 0 10 20 30 40 50 10 20 30 40 0 10 20 30 40 50 advance online publication online advance

Supplementary Table 1 Supplementary

4 CD 5 hCD4 %

cells hCD45 of GFP 4 CD % s cell 5 hCD4 of GFP 4 CD %

+ + + + + + + + loxBTR Transplantation Fig. Fig. CD4+ 6532200 Transduction

HIV-1 RNA copies/ml c

10 10 10 10 10 10 Brec1 treated Not treated CD4+ HIV 1 2 3 4 5 6 3 ) might further increase the potential patient range. the potential increase ) might further loxBTR + ) located within LTRsthe within ) of of located the majority HIV-1 Weeks posttransplantation 1 4 SIN vector CD4+ HIV

6532400 hu-Rag + chr1 4+ 1 0 LV-Brec1 sequence. The sequence. 2 2 Counts: 153 Counts: 194 CD 1 4 6532600 LV-Brec1-GFP 4 LV-Ctr-GFP Position (bp) CD4+ 2 8 or ), indicating that HIV-1 provirus in in HIV-1provirus that indicating ),

8100 3 3 1 , of which 90% are predicted to to predicted are 90% which of ,

8200 HIV-1 (HXB2) 0 20 40 60 80

loxBTR

nature biotechnology nature 4 CD 5 hCD4 %

+ + 8300 loxBTR Ratio: 31 Counts: 47 % cells Ratio: 7% Counts: 14 100

sequence is con also sequence 8400 20 40 60 80 0 % Deep sequencing PBMC s carrying point point carrying s

Average count of fragments per 500 bp

Spleen mappable region of HIV sequence Nontreated 10 20 30 40 50 60 CD4 CD45

Brec1-treated 0 + GF + Liver CD4 n = P + 3 + -

© 2016 Nature America, Inc. All rights reserved. regularly interspaced, short palindromic repeats (CRISPR) and its its and (CRISPR) repeats palindromic short interspaced, regularly (TALENs)nucleases activator–like effector scription or tran the clustered, (ZFN), nucleases finger zinc as such technologies, developed tailored generate to approach enzymes. custom suitable a and versatile evolu very is directed tion that suggests specificities site target different with very recombinases designer Cre-like of generation successful The recombinases Cre-based evolved of sites target recombinase recombinases future of generation properties. new with the accelerating help should Cre/ occurring novel, of naturally discovery the with together information This specificity. for recombinase pinpoint are crucial that acids amino and regions define to us allowed efforts. development drug other to compared period time short a years: 2 to 1 approximately to enzyme new a for time development total the streamlining d, 2–3 within completed be can cycle lution However, one evo is time-consuming. erties prop desired with enzyme a new generating recombine to recom binase Tre evolve to necessary were than the asymmetric recombines specifically and efficiently cycles were required to evolve an enzyme that Cre/ established evolution directed rapid virus escape might not occur. with Brec1 ( did not emerge in patient-derived cells treated time window of up to 21 weeks, resistant HIV-1 600 bars, Scale antigen. p24 HIV and CD3 human for stained and HSC transduced LV-Ctr– either LV-Brec1– or with engrafted mice from presented is prepared sections spleen of ( shown. is LV-Ctr LV-Brec1, and engraftment, each from of ratio human and HIV-1 subsequent for prepared was DNA genomic and HSC, LV-Brec1–transduced or LV-Ctr– either with engrafted were that animals CD4 Human HIV-1 infection. after 24 week at mice ( chart). (bar suspensions single-cell using FACSby necropsy at determined was animal respective the of liver and spleen the (BM), marrow bone PBMC, in cells Human time. over monitored were squares) (blue cells CD45 human of percentage and dots) (red load Viral HIV-1. with infected were pools HSC CD34 blood–derived cord human unselected LV-Brec1–transduced or control) (negative ( CD34 human with engrafted mice NSG 6 Figure nature biotechnology nature offer systems, CRISPR-Cas9 the as known protein, Cas9 associated b a ) Newborn NSG mice engrafted with LV-Ctr– with engrafted mice NSG ) Newborn ) Analysis of antiviral Brec1 activity in NSG NSG in activity Brec1 antiviral of ) Analysis Compared to the directed evolution approach, other recently recently other approach, evolution directed the to Compared The library Brec final the of sequencing Deep Brec1 was generated using substrate-linked + T cells were isolated from the spleen of spleen the from isolated were T cells loxBTR gag

Brec1-mediated antiviral effects in effects antiviral Brec1-mediated c / hbg ) Immunohistochemical analysis analysis ) Immunohistochemical hbg Fig. loxBTR sequences in two animals animals two in sequences -specific real-time PCR. The The PCR. real-time -specific sequence has only remote similarity to similarity remote only has sequence 5 loxP ), supporting the notion that 2 0 site, slightly more cycles more cycles slightly site, starting from the well- the from starting system. 145 generation generation 145 system. loxLTR

loxP advance online publication online advance 1 5 lk rcmiain systems recombination -like . Therefore, Therefore, .

µ + HSC. HSC. m. gag

+

CD4 + -

+

- - -

a HIV-1 RNA copies/ml HIV-1 RNA copies/ml HIV-1 RNA copies/ml 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 loxP b gag/hbg and other and other 9 5 9 5 9 5 1,000 1,500 500

15 22 0 , 20 , Weeks postinfection Weeks postinfection Weeks postinfection 23 , 2 , LV-Ctr 3 1 5 - - .

12 12 12 unpredictable sequence rearrangements after the lesions have been been have lesions the after rearrangements sequence unpredictable to nonhomologous leads pathway this through error-prone Repair pathway. (NHEJ) breaks cellular end-joining the double-strand activate in which results (DSBs), clinical cleavage for Nuclease enzymes these applications. of development the hamper may off-target ture reduce nucleases to programmable made by cleavage been have improvements impressive Although Brec1. for activities off-target similar observe not We did effects. genotoxic other and rearrangements genomic undesired in systems nuclease such for reported been relatively are has cleavage Off-target nucleases drawbacks. have also they generated, rapidly programmable these Although recently. (refs. HIV-1 of LTRs the in sequences (refs. CCR5 co-receptor HIV-1 the targeting nucleases designer several In fact, tools. editing of genome development faster LV-Brec1 15 15 15 20 20 20 c 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 LV-Brec1 LV-Ctr + + + + + + LV-Brec1 % hCD45 CD4 % hCD45 CD4 % hCD45 CD4 50 10 20 30 40 50 10 20 30 40 50 10 20 30 40 0 0 0 PBMC PBM PBMC C BM BM BM 4 1 16– aohr udmna fea fundamental another , 21 1 , Spleen Spleen Spleen 37– 8 ) have been described described been have ) LV-Ctr 4 0 , which may result result may which , ticles e l c i rt A Liver Liver Liver %CD45 %CD4 %CD45 %CD4 %CD45 %CD4 30 , 31 + + + HIV p24 hCD3 GFP GFP GFP , + + + 3 CD4 CD4 CD4 6 ) or or ) + + + + + +  -

© 2016 Nature America, Inc. All rights reserved. believed that future cure strategies will require a combination of of combination a require different will antiviral strategies approaches, cure including future application that of small-molecular-believed strategy antiviral HIV-1 single a of using eradication unachievable is complete the even or in cART) of infection the absence of the control durable (i.e., remission long-term that proviruses. multiple of likely exceeds the risks for unwanted side effects due to recombination activation. Therefore, the benefit for a patient of receiving Brec1 most Brec1-mediated due to oncogene of transformation risk the Thus, reduces excision proviral virus. integrated of copies multiple on nor haploinsufficiency on dependent neither is oncogenes of activation because likely, more much be would insertion HIV-1 through genes tumorigenesis of risk the increase indeed could state haploid a genes, suppressor tumor some for However, phenotype. in change a cause not does genes most for Haploinsufficiency chromosome. functional fully another carry still would cell the that meaning haploinsufficiency, cause would events the consequences are to expected be mild. Virtually all recombination multiple between proviruses would and occur delete genetic material, the of recombination distance Brec1-mediated if even Third, the proviruses. of integrated independent rates, equal at sites target tion recogni the cleave will which CRISPR-Cas), or TALEN ZFN, approach (i.e., nuclease the from system recombinase the distinguishes multiple HIV-1 ( proviruses between recombination of possibility the reducing further proviruses, individual the deleted has recombination when longer any expressed not is Tat, by Brec1 driven is system described the in expression recombinase Because proviruses. integrated rately between recombination than rate higher loxBTR between recombination Therefore, proviruses. of sites individual the two the between distance the than bigger be certainly almost will genome proviruses the of distance the the integrations multiple of in cases rare sites these of distance the with correlates the However, occur. recombination frequency between recombinase target sites negatively theoretically could recombination mediated Brec1- by material genetic of deletion proviruses, HIV-1 integrated more or two carry do that cells In unlikely. highly is proviruses ent HIV-1 of copy DNA one only contain cells infected of majority the that revealed have studies single-cell First, patients. for against risk increased an argue rationales several However, gene. a suppressor deleting tumor example, for potentially, cell, the in sequences genomic recombination of Brec1-mediated proviruses. more) (or two carries considered. is treatment before benefits against balanced carefully be should These risks. bear technologies editing genome other or recombinases site-specific employing therapy gene It that be noted may also approaches prove should editing expedient. genome more or two of combination a and patients, all for effective emphasize that it is not clear whether one system will ultimately prove we However, subjects. HIV-1–infected in proviruses deleting at ing aim applications recombinases clinical future for strategy promising site-specific a represents employing therapy gene Hence, ways. path DNArepair activating without and precision predictable with protein itself, by recombinase the are performed solely The reactions recombinases do not rely on cellular pathways during genome editing. site-specific By contrast, effects. detrimental potential with repaired, s e l c i rt A  strategies modulating immune agents, latency-reversing weight

In recent years, HIV cure research has repeatedly demonstrated demonstrated repeatedly has research cure HIV years, recent In cell a which in case a be would event precarious potentially A loxBTR 42 , sites of one integrated provirus will occur at an exceedingly exceedingly an at occur will provirus integrated one of sites 4 3 . Second, recombination between between recombination . Second, sites from the different proviruses could delete or invert invert or delete could proviruses different the from sites 4 5 . On the other hand, activation of onco of activation hand, other the On . Supplementary Fig. 6 Supplementary loxBTR loxBTR 36 sites of two sepa two of sites ). ). Of note, this fact , s from two differ two s from 46 , loxBTR 4 7 . Rather, it is is it Rather, . 4 4 sites of of sites . In the the In . loxBTR ------

proviral proviral load, it may also improve reconstitution of the functional the the but, by lowering cells), in provirus-containing (i.e., needed when only transgene antiviral the by expressing of biosafety margin a high approaches and kill) shock (e.g., purging Brec1 therapy may HSC-based therefore be ideally combined with cells, novel reservoir Tat-producing in only Brec1 expresses here used vector SIN the As well). equally recognized are viruses dual-tropic sequence target Brec1 the of tion conserva high to due tropism virus of independently occur would process removal DNA. This proviral integrated the remove will tion, is HSC to result in expected HIV-1 these host cell progeny that, upon HIV-1 of infec Engraftment patients. (preconditioned) into gene-modified HSC the of reinfusion autologous by followed be would LV-Brec1(SIN) technology self-inactivating advanced using transfer and factor (G-CSF) upon isolated apheresis. stimulatory CD34 a scenario, such In conceivable. is patients HIV of number small a in 1b/2a) nisms mecha antiviral RNA-based or protein various using trials clinical in tested being currently are approaches transfer gene of types both CD34 or gene-modified CD4 gene-modified antiviral either of transfer autologous therapies gene possibly and online Note: Any Supplementary Information and Source Data files are available in the PRJNA28774 codes: accession BioProject following the under codes. Accession the t of in version available are references associated any and Methods M be directly injected into patients to deliver Brec1 transiently to CCR7 cell tailored type–specific ankyrin repeat proteins (DARPins) displaying (AAV) vectors, viral nonintegrating adeno-associated example, engineered For capsid future. near the in may this developments allow vector indeed new fact, In desirable. highly be cells would reservoir to Brec1 of targeting direct infection, latent to eliminating respect with Therefore, reached. may be cell reservoir infected latently every not approaches combinatory advanced such CD4 peripheral adult the target simultaneously example, for that, strategies with or treatments immune-modulatory with combined be also could apy technology. Brec1 from benefit greatly may cells infected latently remaining of killing quent subse and purging drug-induced Indeed, system. patient’simmune by the DFG (BU 1400/7-1), the BMBF (GO-Bio FKZ 0315090), the Else for next-generation sequencing of nrLAM-PCR products. This work was supported for technical assistance, and D. Indenbirken and M. Alawi (Heinrich Pette Institute) construction, G. Pilnitz-Stolze, B. Weseloh and B. Abel (Heinrich Pette Institute) for DNA sequencing, D. Dubrau (Heinrich Pette Institute) for initial ASLV-vector CBG) and A. Dahl (DFG-Forschungszentrum für Regenerative Therapien Dresden) and Genetics; MPI-CBG) for the generation of transgenic mice, S. Winkler (MPI- for SKY analyses, R. Naumann (Max Planck Institute for Molecular Cell Biology reagents, R. Kuhnert (TUD) for help with mouse work, A. Käßner-Frensel (TUD) The authors are indebted to M. Karimova (TU Dresden; TUD) for providing HIV-1 for (ref. reservoir cellular major a represent to shown (Tcm) CD4 memory central A ckn ethods Besides latency-reversing agents, Brec1-based stem cell gene ther gene cell stem Brec1-based agents, latency-reversing Besides version of the pape ow 5 0 . . With respect to Brec1, an HSC-based phase 1 trial (i.e., phase advance online publication online advance ledgments he pape he 7 . + + HSC would be mobilized with granulocyte-colony granulocyte-colony with mobilized be would HSC T cells with antiviral entities. However, even with with even However, entities. antiviral with cells T Sequencing data are available from the NCBI SRA SRA NCBI the from available are data Sequencing r . r . + HSC into HIV-infected subjects HIV-infected into HSC + 1 T cells, a cell population that has been been has that population cell a cells, T , 4 8 . Gene therapies might employ the the employ might therapies Gene . loxBTR

51 (i.e., CCR5-, CXCR4- and and CXCR4- CCR5-, (i.e., nature biotechnology nature , 5 2 . This not only provides . not provides only This PRJNA28845 Ex Ex vivo 4

9 . Indeed, . Indeed, + 5 3 T cells cells T 5 in vivo in , , could online online 4 8 gene ). and and

+ - - - - -

© 2016 Nature America, Inc. All rights reserved. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm R version of t The authors declare competing interests:financial details are available in the wrote the manuscript. reagents. J.H. and F.B. the designed study, directed the study, evaluated data and analyses and structure-based evaluated data. A.S., C.L. and J.v.L. provided essential E.S. experiments designed and evaluated data. J.A.-G. and M.T.P. performed experiments, designed performed computational analyses and evaluated data. experiments, performed experiments and evaluated data. A.G., M.P.-R. and V.S. and wrote the manuscript. C.S., D.C., N.B., H.H.-S., U.C.L. and K.H. designed J.K., I.H. and J.C. experiments, designed performed experiments evaluated data IRES-puro was a generous gift from K. Anastassiadis (TUD). Regenerative Therapien Dresden). The mammalian expression vector pCAGGS- EGFP gene, was a generous gift from E.M. Tanaka (DFG-Forschungszentrum für mCherry gene was inserted between the CAG enhancer/promoter module and the The reporter plasmid pCAGGS- Heinrich Pette Institute–Leibniz Institute for Experimental Virology, Hamburg. Kröner-Fresenius-Stiftung (2010_A82), and the “Viral Latency” program at the nature biotechnology nature C A eprints and permissions information is available online at at online available is information permissions and eprints uth O

MPETING FINANCIAL INTERESTS FINANCIAL MPETING Santoro, S.W. & Schultz, P.G. Directed evolution of the site specificity of Cre of specificity site the of evolution Directed P.G. Schultz, by & S.W. specificity Santoro, site recombinase Cre of Alteration A.F. Stewart, & F. Buchholz, B.H. Habermann, & F. Buchholz, J., Hauber, J., Chusainow, V., Surendranath, W.Hu, CRISPR/Cas9 the Y.Harnessing Koyanagi, Y.& Kanemura, N., Misawa, H., Ebina, X. Qu, using excision F.DNA Buchholz, proviral HIV-1& J. Hauber, I., Hauber, I., Sarkar, L.M. Beltrán, B.R. Schackman, M. Rappold, J.B. Nachega, Petoumenos, K. L. Pinoges, resistance. drug Antiviral D.D. Richman, Akay,C. of Re-emergence A.S. Fauci, & H.C. Lane, D., Engel, T.W.,Jr.,R.T.Davey, Chun, mechanisms molecular of update an latency: HIV-1 M. Sgarbanti, & A. Battistini, signaling cellular latency: HIV of control Transcriptional J. Karn, & U. Mbonye, and discovery,feasibility, cure: HIV An G.K. Folkers, & H.D. Marston, A.S., Fauci, pathogenesis, epidemiology, infection: HIV S.R. Lewin, & C. Celum, G., Maartens, Passaes, C.P. & Sáez-Cirión, A. HIV cure research: advances and prospects. recombinase. evolution. protein substrate-linked systems. recombination (2010). site-specific W293–W298 directed of and detection the for evolution tool search site recombination of locus SeLOX--a infection. HIV-1 new provirus. HIV-1 latent disrupt to system 41 cells. T human infected latently and infected from DNA proviral recombinase. evolved an Manag. Risk cardiovascular risk associated with the human immunodeficiency virus. States. United the in care (2014). 19768 2014. and 2011 between therapy antiretroviral concepts. evolving study. (D:A:D) Drugs Anti-HIV of Events Adverse on Collection Data the patients: HIV-positive in analyses. longitudinal and cross-sectional Syndr. Defic. Immune Acquir. J. multicentric therapy: antiretroviral system. nervous central the therapy. stopping after HIV strategies. therapeutic and (2014). 328–339 pathways, epigenetics, happenstance and the hope for a cure. a for hope the and happenstance epigenetics, pathways, implementation. prevention. and treatment, 454–455 , 7771–7782 (2013). 7771–7782 , o r et al. et Co et al. et he pape et al. et , 340–352 (2014). 340–352 , ntributi et al. et Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 excision efficient and specific mediate Zinc-finger-nucleases

RNA-directed gene editing specifically eradicates latent and prevents and latent eradicates specifically editing gene RNA-directed 11 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. Antiretroviral drugs induce oxidative stress and neuronal damage in damage neuronal and stress oxidative induce drugs Antiretroviral et al. et t al. et J. Int. AIDS Soc. AIDS Int. J. l t al. et . r , 35–48 (2015). 35–48 , et al. . J. Am. Med. Assoc. Med. Am. J. Risk factors and mortality associated with resistance to first-line to resistance with associated mortality and factors Risk et al. et Infect. Disord. Drug TargetsDrug Disord. Infect. Influence of immune activation and inflammatory response on response inflammatory and activation immune of Influence Treatment modification in HIV-Infected individuals starting individuals HIV-Infected in modification Treatment I tetet deec, rg eitne vrlgc failure: virologic resistance, drug adherence, treatment HIV D:A:D Study Group. Predicting the short-term risk of diabetes Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. The lifetime cost of current human immunodeficiency virus immunodeficiency human current of cost lifetime The o Science ns Med. Care Med. Lancet Viruses Nature J. Neurovirol. J. loxP

advance online publication online advance

15 68

-mCherry- 316

Nat. Biotechnol. Nat. 384 401 6 , 17426 (2012). 17426 , , 527–535 (2015). 527–535 ,

, 1715–1758 (2014). 1715–1758 ,

312 44 , 1912–1915 (2007). 1912–1915 , , 258–271 (2014). 258–271 , , 874–875 (1999). 874–875 , Sci. Rep. Sci. , 990–997 (2006). 990–997 ,

Antiviral Res. Antiviral , 335–336 (2014). 335–336 ,

99 20 loxP , 4185–4190 (2002). 4185–4190 , , 39–53 (2014). 39–53 ,

11

-EGFP, where a floxed . n. IS Soc. AIDS Int. J. 111

, 167–174 (2011). 167–174 , 3

19 , 2510 (2013). 2510 , , 11461–11466 (2014). 11461–11466 ,

, 1047–1052 (2001). 1047–1052 , uli Ais Res. Acids Nucleic 71 http://www. , 117–121 (2006). 117–121 , Nucleic Acids Res. Acids Nucleic Virology

17 nature.com/ Vasc. Health spl 3), (suppl.

454–455

Virology online

38 , ,

34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 24. 23. 22. 54. 53. 52. 51. 43. 42. 41. 50. 49. 48. 47. 46. 45. 44. 40. 39. 38. 37. 36. 35.

rwl, T.A. Crowell, challenge The S.M. Hammer, F.E.& McCutchan, M.E., Sobieszczyk, Taylor,B.S., J.B. Whitney, N. Holt, E.E. Perez, I. Hauber, Self-inactivating A. Schambach, & C. Baum, M., Galla, T., Maetzig, J.D., Suerth, of structure Crystal D. F.,A.F.Suck, Buchholz, Stewart, & Meyer,J.E., Ennifar,E., cre- of view structural A G.D. Duyne, Van J. Abi-Ghanem, LTR-specific HIV-1 of analysis and evolution vitro In J. Hauber, & F. Buchholz, M. Karimova, homolog Cre heterospecific a with recombination DNA J. McDermott, & B. Sauer, Chomont, N. Chomont, R.C. Münch, C. Katlama, kill. and Shock HIV: S.G. Deeks, L. Josefsson, Josefsson, L. nucleases. programmable with engineering genome to guide A J.S. Kim, & H. Kim, Herrera-Carrillo, E. & Berkhout, B. Bone Marrow Gene Therapy for HIV/AIDS. treatment. HIV-1 for therapy gene vivo Ex J.J. Rossi, & L.J. Scherer, Blankson, J.N., Siliciano, J.D. & Siliciano, R.F. Finding M. aCaskey, cure for human immunodeficiency N.M. Archin, Ashworth,HaploinsufficiencySantarosa,A.& M. tumour suppressor for genes: when Ringrose, L., Chabanis, S., Angrand, P.O., Woodroofe, C. & Stewart, A.F. Quantitative X. Wang, Tsai,S.Q. R. Gabriel, cleavage off-target Revealing D.R. Liu, & V.,J.K. Pattanayak, Joung, C.L., Ramirez, Tebas,P. Suzuki, E. & Nakayama, M. VCre/VloxP and SCre/SloxP: new site-specific recombination n pros ih eial cnrle HV infection. HIV controlled medically with persons and diversity. subtype HIV-1 of monkeys. rhesus (2010). HIV-1 control CCR5 to targeted nucleases nucleases. zinc-finger using editing mice. humanized in recombinase design. (2010). split-packaging a with vectors alpharetroviral Res. activation. cleavage DNA for mechanism alternative an suggesting recombinase- Cre wild-type a Struct. Biomol. Biophys. Rev. approach. (2013). structure-guided 2394–2403 and evolution- combined recombinases. system. recombination phages. ofP1-related Res. Acids regions the pac-c1 of comparison from identified and homeostatic proliferation. homeostatic and vectors. viral reservoirs. HIV-1 7 (2011). 11199–11204 Genet. Rev. Nat. (2015). Genet. virus-1infection. 3BNC117. antibody neutralizing therapy. antiretroviral on way.theall go toneeddon’t you distances. short at flexibility DNA effective increases chromatin vivo: in and vitro in looping DNA of comparison Pathog. PLoS T-cellsCD4+ of majority the that reveals HIV-1patients one contain molecule. DNA molecule. DNA HIV one only contain individuals vectors. lentiviral integrase-defective TALENsusing nucleases. CRISPR-Cas specificity. (2011). specificities of zinc-finger nucleases by HIV.with engineering.genomesystemsfor (2015). 1692–1702 , 3910–3936 (2015). 3910–3936 ,

31

20 , 5449–5460 (2003). 5449–5460 , et al. et et al. et N. Engl. J. Med. J. Engl. N. , R100–R107 (2011). R100–R107 , et al. et t al. et

Nat. Biotechnol. Nat. 32 t al. et t al. et t al. et t al. et et al. et t al. et et al.

Nat. Commun. Nat. et al. et t al. et et al. et et al. et et al. et Human hematopoietic stem/progenitor cells modified by zinc-finger by modified cells stem/progenitor hematopoietic Human , 6086–6095 (2004). 6086–6095 , 9 t al. et Gene editing of CCR5 in autologous CD4 T cells of persons infected persons of cells T CD4 autologous in CCR5 of editing Gene Methods GUIDE-seq enables genome-wide profiling of off-target cleavage by cleavage off-target of profiling genome-wide enables GUIDE-seq , e1003432 (2013). e1003432 , nisd eeto o oftre cevg b CIP-a9 and CRISPR-Cas9 by cleavage off-target of detection Unbiased

Infect.Dis.Clin. North Am. Lancet Nature t al. et Highly significant antiviral activity of HIV-1 LTR-specific tre- LTR-specific of HIV-1 activity antiviral significant Highly 15 salsmn o HV1 eitne n D+ cls y genome by cells T CD4+ in resistance HIV-1 of Establishment Off-target-free gene delivery by affinity-purified receptor-targetedaffinity-purified by delivery gene Off-target-free Majority of CD4+ T cells from peripheral blood of HIV-1-infected ares o cr fr I: e wy t tre ad eradicate and target to ways new HIV: for cure a to Barriers iama upesd n I--netd uas y broadly by humans HIV-1-infected in suppressed Viraemia HIV reservoir size and persistence are driven by T cell survival cell T by driven are persistence and size reservoir HIV An unbiased genome-wide analysis of zinc-finger nuclease zinc-finger of analysis genome-wide unbiased An Vika/vox, a novel efficient and specific Cre/ specific and efficient novel a Vika/vox, Administration of vorinostat disrupts HIV-1 latency in patients in latency HIV-1 disrupts vorinostat of Administration optlzto rts n raos mn HV lt controllers elite HIV among reasons and rates Hospitalization Single cell analysis of lymph node tissue from HIV-1 infected HIV-1 from tissue node lymph of analysis cell Single ai seig f h vrl eevi pir o I vrei in viraemia SIV to prior reservoir viral the of seeding Rapid , 321–334 (2014). 321–334 , Engineering of a target site-specific recombinase by a recombinase site-specific target a of Engineering Nucleic Acids Res. Acids Nucleic

Nat. Biotechnol. Nat. 53 512 381 Nature

, 102–109 (2011). 102–109 , N. Engl. J. Med. J. Engl. N. 370

29 6 , 74–77 (2014). 74–77 , , 2109–2117 (2013). 2109–2117 , EMBO J. EMBO

, 6246 (2015). 6246 , loxP 30 Nat. Med. Nat. , 816–823 (2011). 816–823 , , 901–910 (2014). 901–910 ,

487 Nucleic Acids Res.NucleicAcids Biochim.Biophys.Acta , 87–104 (2001). 87–104 , Nature Nature PLoS Pathog. PLoS yas rvas nvl pcr conformation spacer novel a reveals synapse Nat. Biotechnol. Nat. , 482–485 (2012). 482–485 ,

in vitro 18

522 487 33 , 6630–6641 (1999). 6630–6641 , 15

loxP

in vivo in 41 28

, 187–197 (2015). 187–197 , , 893–900 (2009). 893–900 , , 487–491 (2015). 487–491 , 358 , 439–440 (2012). 439–440 , selection. , e37 (2013). e37 , , 633–650, (2014). site-specific recombination. site-specific

9 , 1590–1602 (2008). 1590–1602 , Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. . , e1003587 (2013). e1003587 , Nat. Biotechnol. Nat.

Nat. Biotechnol. Nat. 39

26 , e49 (2011).e49 ,

. Virol. J. , 808–816 (2008). 808–816 , Nat. Methods 1654 uli Ais Res. Acids Nucleic ticles e l c i rt A . net Dis. Infect. J. loxP , 105–122(2004).,

84 -like site-specific -like

6626–6635 , uli Acids Nucleic 28 33

8 , 839–847 , , 175–178 , u. Mol. Hum. , 765–770 Nucleic Viruses

Annu.

108 211 41  , , ,

© 2016 Nature America, Inc. All rights reserved. ing sequences that had successfully recombined the vector were subsequently were subsequently the vector recombined that had successfully ing sequences cod recombinase The step. amplification PCR following the for unsuitable them that NdeI had the rendering plasmids not recombined, to been linearize with digested and culture overnight induced an from isolated DNAwas mid plas For cycle, each at levels. lower expression activity with for recombinases l 200 with induced was expression recombinase cycle evolution first For the levels. of expression E. coli previously as described ( to substrate information the protein the coding linking thereby sequence, target recombinase the contained also that plasmid a pEVO-recomb-target, vector evolution the into cloned was library binase Tosequence. protein evolution, the substrate-linked complex perform recom such a way that they best resembled Cre without resulting in an altered protein in sequence nucleotide the changing by (“Cre-ed”) modified were Shew and number sion N namely Dre recombinases, (Cre/Fre) Cre-like mutants Cre known of library a several and Cre and pooling shuffling by family generated was recombinases mutated randomly of strategy. library evolution and libraries recombinase of Generation (NEB). BioLabs England New from purchased were enzymes restriction localization signal (NLS) before ligating it into the above vectors. described All and the with equipped was Brec1 a sequence codon-optimized coding nuclear Anastassiadis) Konstantinos from gift (a generous pCAGGS-IRES-puro vector expression the mammalian methods. cloning the pCAGGS- Dresden). EGFP were from generated pCAGGS- Therapien loxLTR Regenerative für Forschungszentrum enhancer/promoter Tanaka (DFG- M. Elly from CAG gift generous a was the gene, EGFP the and module between inserted was gene mCherry floxed vector. the into it ligating before sion in mammalian cells and equipped with a nuclear localization signal (NLS) expres for codon-optimized was sequence coding Brec1 The pIRESneo-Tre. and pIRESneo-Cre for described as manner same the in generated was Brec1 and tors pIRESneo-Tre pIRESneo-Cre were previously reported and the vector restriction sites AgeI and NruI. The mammalian expression vec CGACCATGGTGGCCCGG- AGCTTGCATGCCTGCAGGTC-3 loxBTR with sites previously pSV and pSVpaX plasmids reporter multi-host The (5 AACAATTAA-lox-TAGACTGGATGGAGGCGGATAA-3 (5 lox2-F primers the used step cloning second The TCGACTGTTCCTA-3 TCTCAGTACAATCTGCTCTGATGC-3 (5 lox1-F primers the ing step used (System following Biosciences) the manufacturer’s The instructions. first clon pEVO- from steps cloning previously pEVO- described as methods cloning standard using generated were sites target and recombinases of sequences coding described the containing pEVO- and (pEVO-6) assays. reporter and expression recombinase evolution, protein substrate-linked for Plasmids METHODS ONLINE nature biotechnology nature by P1 PCR amplified the (5 using primers -arabinose -arabinose concentration cycle by cycle (to 1 Z ′ The The multicycle substrate-linked protein evolution (SLiPE) process was done into sequence coding Brec1 the subcloning by built was pCAGGS-Brec1 pCAGGS- plasmid reporter The -ACTTCGGGCTCATGAGCC TAGA-3-ACTTCGGGCTCATGAGCC _ loxP 2 A 2 , Zre (isolated from from (isolated Zre , under the control of an inducible promoter (P promoter of control the an inducible under B loxBTR -mCherry- F (5 -F E sites with either W 1 loxBTR 0 5 . pSV . 1 C ′ 0 CTCTATAACGTCG TTCGA- -CCTACTTCACTAACAACCGGTACAG - 0 P loxP 0 0 0 0 loxBTR sequences using Cold Fusion Cloning with the primers primers the with Cloning Fusion Cold using sequences 1 µ loxLTR 0 5 g/ml of of g/ml n pEVO- and 4 ), and Shew ( 7 loxLTR 0 15 h eouin etr pEVO- vectors evolution The ′ ). To promote the efficiency of family shuffling, Dre Dre shuffling, family of efficiency the Topromote ). loxLTR ) and the restriction enzymes ApaLI and AvrII. AvrII. and ApaLI enzymes restriction the and ) , was generated from pSVpaX by replacing the the replacing by pSVpaX from generated was 2 EF ad pCAGGS- and -EGFP loxBTR 4 Salmonella enterica Salmonella . Briefly, the recombinase library was expressed in was expressed library . Briefly, the recombinase l 5 -arabinose (Sigma-Aldrich). By decreasing the the decreasing By (Sigma-Aldrich). -arabinose loxBTR 5 were described previously described were . For expression in mammalian cells or mice, mice, or cells mammalian in expression For . sequences or Shewanella loxBTR using the Cold Fusion Cloning technology technology Cloning Fusion Cold the using ′ -CTTAGATCCTCATATGGTGCAC-lox- ′ 2 ) and and ) -GGATCCAGACATGATAAG ATAC-3 0 loxP , a previously generated Tre library Tre generated previously a , loxP - ′ loxH ′ -CTCTACTGTTTCTCCATAC-3 -mCherry- ) and lox1-R (5 lox1-R and ) loxBTR ′ -mCherry- ) and the restriction enzyme ) AseI. enzyme and the restriction sp. strain ANA-3, GenBank acces loxBTR µ were each generated in two two in generated each were g/ml), g/ml), the library was enriched , GenBank accession number number accession GenBank , Supplementary Fig. 1b Fig. Supplementary loxBTR -R (5 -R sequences using standard BAD loxP loxLTR ′ -CTAGCTTCCCGGC loxP ′ -CCAAGCTACTCG ) allowing regulation regulation ) allowing -mCherry- 15 -EGFP by -EGFP replacing loxP ′ , -TGCAGTGTCG -TGCAGTGTCG EF, hr a where -EGFP, 2 ′ 0 were described described were ) and lox2-R lox2-R and ) . pEVO vectors vectors pEVO . pEVO- , 1 5 A complex complex A . . pIRESneo- loxBTR loxBTR ). loxH loxP 1 1 ′ 5 5 ′ ------) ) . ,

induction of recombinase expression, recombination on recombination expression, of recombinase induction alternating cycles more by followed cycle, second the loxBTR vector containing the two against ( Brec to specificity enhance alternately formed on activity recombination against selecting cycles Brec a Once library with high activity on the mutations. advantageous combine to shuffled were they sites, target subset the on activity high with recombinases for libraries subset the site target final on the steps cycling evolution for the 1 and from 2 subsites libraries the and combining shuffling when was applied Asymmetry complement. reverse the being site half other the and mutations the required one with site sites, half target containing as symmetric generated ( nucleotides differing few a only ( sites target subset on initialized first P2 and (5 above) (see P1 primers the using coding PCR by amplified recombinase were sequences full-length the shuffling, DNA Following mutations. previously described as out carried was shuffling DNA cycle, next evolution third the each After for cycle. vector evolution evolution the into back subcloned and XbaI and BsrGI with digested was product PCR The cycle. every in library recombinase the of variability increase and mutations more generate to used was polymerase (5 P3 and Ion Sphere Particles (ISPs) using the Ion OneTouch 200 Template Kit v2 DL DL v2 Template Kit 200 OneTouch Ion the using (ISPs) Particles Sphere Ion OneTouch200 Ion template-positive preparing by sequencing for processed further was library the Technologies), (Life Kit Quantitation Library Ion using the quantification assay TaqMan Following Technologies). (Life gel rose aga 2% SizeSelect E-Gel an with size-selected was library the Technologies), (Life Kit Library Fragment Plus Ion the using PCR by amplification and ers adapt of ligation After (Qiagen). Kit Purification PCR QIAquick the using aiming for instructions, 200-bp The fragments. DNAfragmented was purified manufacturer’s the following Technologies) Kit (Life Ion Reagents Plus Shear tive agarose gel. Subsequently, the DNA was fragmented enzymatically using the library and purifying the linear 1-kb-sized recombinase library from a preparaplasmid pEVO-recomb-target of digestion XbaI and BsrGI by prepared was using the Ion Torrent semiconductor system. Whole library recombinase DNA sequencing. Next-generation specificity. site target and efficiency in analyzed and isolated were clones vidual indi hundred a than More clones. recombinase individual of selection and by PCR and subcloned back into the pEVO- following the manufacturer’s The Brec final was library retrieved instructions. (Qiagen) Kit Mini Blood DNA QIAamp the using extracted DNA genomic and harvested were recombinases well-tolerated expressing apparently cells sorting, final the following culturing of d 4 another After expression. EGFP (FACS) on days 19 and 29 after transduction with increased sorting stringency forcell high fluorescence-activated sequential by expression EGFP for sorted were cells T Jurkat Transduced cells. human in of EGFP and co-expression recombinase the constitutive for below) (described vector lentiviral a into subcloned was cycles 145 after obtained library Brec active the cells, malian for recombinases that are well tolerated even at high expression levels in mam screening for Cellular well-tolerated recombinases in human cells. loxBTR against against cycles three (i.e., selection counter of cycle sixth each after out carried was shuffling DNA cycle. evolution next for the vector evolution the into back subcloned and above described as PCR recombined successfully that had sequences coding NdeI and with recombinase the was digested cycle 1 to down cycle by cycle not. on recombination whereas site, NdeI single the of removal ′ The evolution procedure was carried out in a stepwise manner and was was and manner stepwise a in out carried was procedure evolution The -AATCTTCTCTCATCCGC-3 l -arabinose was applied at 100 applied was -arabinose for one more, final cycle. final more, one for - loxP loxP loxH ′ and and . h Be lbay band a sblnd ak no pEVO- into back subcloned was obtained library Brec The ). -AGGGAATAAGGGCGACA-3 ) for the first cycle and with with and cycle first the for ) loxH , the evolved Brec library was cloned into an evolution evolution an into cloned was library Brec evolved the , loxBTR loxBTR µ g/ml. Plasmid DNA isolated after each evolution evolution each after isolated DNA Plasmid g/ml. 15 , 2 Evolved recombinase libraries were sequenced were sequenced libraries Evolved recombinase 4 loxBTR to rapidly propagate and combine beneficial beneficial combine and propagate rapidly to sites intertwined with two rather than than rather ′ Supplementary Fig. 1a Fig. Supplementary ). µ g/ml during the first cycles and reduced and reduced cycles first the during g/ml Fig. 2 Fig. site was obtained, additional evolution loxP loxH loxBTR a ′ ) with each of them containing containing them of each with ) loxH E. coli E. ) sites (pEVO- sites Fig. Fig. 2 15 alternating with three cycles cycles three with alternating , 2 4 or or vector for analyses further Te ro-rn Taq error-prone The . loxP for their recombination recombination their for a loxBTR loxP ). For counter selection ). For counter selection loxH and and loxBTR doi: ). All subsites were were subsites All ). were amplified by amplified were loxH loxH loxBTR 10.1038/nbt.3467 and and . After enriching enriching . After loxH results in results the sites (pEVO- or or loxP were per were - loxP To select loxP . Upon . does does ) for for ) ------

© 2016 Nature America, Inc. All rights reserved. as described above. Recombinase expression was induced with 1 mg/ml on mg/ml 1 with induced was l expression Recombinase in tested above. was sites described as variant the instructions. on Brec1 manufacturer’s of activity the Recombination following (Stratagene) Kit Mutagenesis pEVO-Brec- of mutagenesis ( database gov sequence HIV Alamos Los the using tified with HIV-1 strains on activity Brec1 for Assay plasmid. reporter the for selecting plates again plated into were on Cells cells. subsequently XL1-Blue X-gal-containing was site target nonspecific a for chosen DNAPlasmid the expression plasmid. plasmids, was and isolated transfected two the of sites target the between competition avoid To (chloramphenicol). plasmid expression recombinase mg/ml 1 at recombinase expression plasmids (pEVO-recomb-target) and grown overnight pSV plasmids reporter the on activity its to compared and cells) mammalian in delivery Brec1 for designed vector loxP gel. agarose an on substrate nonrecombined versus recombined for sizes fragment different in resulting (NEB), XbaI and BsrGI with digested was culture overnight induced an from recomb-target) of efficiency recombinase libraries or individual clones, plasmid DNA (pEVO- sion was induced with 0–1,000 Recombination assays and tests specificity in bacteria. (Cre/ of systems three the each in mutated differently residues those to attention particular detail paying in analyzed and found were regions hotspot mutational Four Cre. of of sequences Tre and the clones obtained in the Brec with the library sequence mutations observed Cre/ of the ture mutations. of analyses Structure-based allowed. were mismatches ten to up ‘N’s, with additionally, and replaced was part spacer the search, For the loxBTR to similar sequences resembling of for sequences presence the searched tively exhaus were genome human the to align properly to failed that reads full all number accession the ( BioProject SRA NCBI the statistics were in calculated R version (ref. 3.1.2. Sequencing data are available 1.2.1 from version samtools by handled was processing files output Bowtie2 reversed. were steps alignment two last HIV, the from originating reads of set a Tocreate sequence. recombinase the from nating origi unambiguously fragments of set a formed reads mapped Successfully in the mutations not did reveal data sequencing deep the of Inspection provirus. expression recombinase the of ments) and all reads that remained unmapped were then to aligned a sequence ( sequences HIV of database a to setting) option “–sensitive” a (with mapped then were align to failed that “–no-mixed,” read pairs All “–no-discordant,” settings. and “–fast” parameter (ref. 2.1.0 version bowtie2 control-treated using and platform. Brec1-treated 2000 from HiSeq samples were initially aligned to the obtained human reference genome Illumina (hg38 assembly) reads an sequencing using The sequencing deep by analyzed chip. Ion an 314 and system Ion Kit with the Ion PGM 200 Sequencing (PGM) Machine Genome Personal the using sequenced was library the Finally, System. OneTouch Ion the with doi: -arabinose and recombination assayed as described above. Recombination Recombination above. described as assayed recombination and -arabinose The lacZ-based recombination reporter assay used XL1-Blue cells harboring To test specificity, substrate Brec1 was recombinase tested for onits activity expression, Brec1 by caused rearrangements genomic potential Todetect was patient HIV-1-infected an of cells T from isolated DNA Genomic loxBTR / 10.1038/nbt.3467 , , ). The respective respective The ). loxLTR . This step was performed with patman aligner version 1.2.2 (ref. (ref. 1.2.2 version aligner patman with performed was step This . served as a positive control. positive a as served loxBTR l and SIN and -arabinose, selecting for both the reporter (ampicillin) and the the and (ampicillin) reporter the both for selecting -arabinose, loxP . loxBTR complex 2 5 ∆ loxBTR . Jalview . PRJNA28774 lox ( lox loxP -like sites as potential targets for Brec1 were iden were Brec1 for targets potential as sites -like loxBTR loxBTR , Tre/, http://ww 26 -like target sites were generated by site-directed site-directed by generated were sites target -like loxBTR loxBTR http://www.hiv.lanl µ , 5 2 9 7 g/ml g/ml was used to prepare the alignments of the the of alignments the prepare to used was (PDB ID ID (PDB loxLTR 5 6 , pSVpaX or pSV or pSVpaX , -like site present in a lentiviral expression expression lentiviral a in present site -like ). A local alignment mode was used with with used was mode alignment local A ). 7 loxBRT using the QuickChange Site-Directed Site-Directed QuickChange the using . l -like sites from viral strain variants. strain viral from sites -like w.ncbi.nlm.nih.gov/b -arabinose. -arabinose. To assay the recombination , Brec1/ , The high-resolution crystal struc crystal high-resolution The 1Q3 site for the Brec1-treated sample. sample. for site Brec1-treated the U loxBTR ) was used to map in 3D the the 3D map in to used ) was .gov loxLTR / , “Filtered web” align “Filtered , ). Recombinase expres h 5 ttp://www.hiv.lanl. 7 , transfected with with transfected , ioproject ) and read count count read and ) / ) under under ) . coli E. 5 8 ). ). ------

sion of EGFP that was silent before recombination ( recombination before silent was that EGFP of sion and hence excised upon recombination, allowing the promoter to drive expres sites target recombination two the between sandwiched was sequence coding mCherry The promoter. same the by driven both plasmid, recombined the from expressed (EGFP) protein fluorescent green and enhanced recombined, non the from expressed constitutively being (mCherry) protein fluorescent and red with exclusively Cherry monomeric plasmids reporter plasmids double-fluorescent expression recombinase with co-transfected were cells HeLa early immediate promoter/enhancer. major For recombination the reporter assay, fluorescence-based (CMV) cytomegalovirus human the from Brec1 expression of high-level constitutive for pIRESneo, vector, expression malian mam a into subcloned was culture. sequence cell coding NLS-Brec1 mammalian in Codon-optimized tests specificity and assays Recombination at 5 (Sigma-Aldrich) puromycin with 2 loxLTR transfection. after h 24 medium selective into passaged were cells transfection, stable For instructions. manufacturer’s before detail in described as spinoculation to subjected were cultures the and Technologies) (STEMCELL CC110 StemSpan cocktail cytokine the with prestimulated were cultures HSC For transduction, system. RoboSep and the the EasySep human cord blood CD34 RNA copies/ml). (48,500–280,000 range intermediate the in were therapy starting was 330 cells/ before count CD4 nadir study. Median this in blood donating before study 3 phase in a clinical 3 Pt. participated TDF/FTC/ATZ/r). pt.3 TDF/FTC/MVC; stable cART for more than 5 years (range: 5–13 years: pt.1 TDF/FTC/EFV; pt.2 received had and years) 52–70 (age: male were cART.patients All long-term were as follows: previously as spinoculated were described cells the and (Sigma-Aldrich) polybrene 2 of presence the in added were instructions. particles manufacturer’s viral the pre-stimulation, After to according h 24 for magnetic (Invitrogen) CD3/CD28 beads with pre-stimulated were cultures transduction, For mM 2 RPMI penicillin-streptomycin, in U/ml 100 cultured with FCS, and 10% with supplemented Technologies) 1640 STEMCELL kit, enrichment cell T CD4 (Human instructions manufacturer’s the per as beads magnetic using conditions. culture mycoplasma-free ensure to used was PCR Multiplex analyses. STR and PCR multiplex by authenticated were lines Cell PCR. multiplex by contamination mycoplasma of absence for screened were lines cell All centrifugation. dient gra hyperdensity by cells intact from separated were cells T PM1 dead week Technologies). Life Once a (Invitrogen and penicillin-streptomycin 100 U/ml medium (Lonza) supplemented with 10% FCS, glutamate, pyruvate (Biochrom) at cultured 37 °C and 5% CO were cells T 3038) Number Catalog Program, Reagent Reference & Research Technologies). Life (Invitrogen penicillin-streptomycin U/ml 100 and (FCS) FBS 10% with supplemented pyruvate) GlutaMAX, glucose, (high Medium CO 5% and °C 37 at monolayers as cultured cells HIV-1–infected and CCL-2) Number Catalog (ATCC, wild-type Cell culture conditions, transfection and reporter cell line generation. viral viral blips) and stable immune reconstitution with CD4 counts >500/ confirmed no copies/ml, RNA 50 below load viral years (>5 replication viral of control on long-term based selected were and years) 13–21 range years, 18 duration (median infection HIV-1 established chronic had patients All tion. replica HIV of control long-term with patients three of total a from isolated GFP (23% GFP 3 × 10 µ Reporter HeLa cell lines with stably integrated target sites sites target integrated stably with lines cell HeLa Reporter the to according (QIAGEN) Effectene using transfected were cells HeLa of Preparation CD34 ain-eie HV1ifce CD4 HIV-1–infected Patient-derived Jurkat (ATCC, clone E6-1, Number Catalog and TIB-152) PM1 (NIH AIDS g of reporter plasmid pSVloxBTR, pSVpaX or pSV or pSVpaX pSVloxBTR, plasmid reporter of g + ) and 1 × 10 × 1 and ) 6 CD4 l were generated by HeLa cells grown transfecting in 90 mm dishes with -glutamine and 50 U/ml recombinant human IL-2 (Sigma-Aldrich). (Sigma-Aldrich). IL-2 human recombinant U/ml 50 and -glutamine 2 + 9 ) and 1 × 10 × 1 and ) + . . Transduction rates for CD4 T cells (30% GFP Figure 5 6 CD4 + 6 + d (25% GFP (25% HSC from umbilical cord blood was performed with cord was HSC performed from blood umbilical T cells (18% GFP (18% cells T : LV-Ctr; 2 × 10 2 + at a of density 1 × 10 µ ); l l (range: cells/ 328–516 Supplementary Figure 7 + + ) CD4 ) selection kit (STEMCELL Technologies) µ g/ml. + 6 CD4 T cells transplantation experiments experiments T transplantation cells + T cells were negatively selected selected negatively were cells T + + T cells, respectively. T cells were were cells T respectively. cells, T ), respectively; LV-Brec1: 10 × 1 respectively; ), 2 + in Dulbecco’s Modified Eagle Eagle Modified Dulbecco’s in nature biotechnology nature T cells (42% GFP 6 cells per ml in RPMI 1640 Fig. 4 Fig. µ loxLTR l) l) and viral loads peak : LV-Ctr; 1 × 10 a ). HeLa cells were were cells HeLa ). loxBTR , and selection selection and , + ), LV-Brec1; µ , , l l during 2 loxP 6 9 µ HeLa (25% were were g/ml g/ml 2 9 or . + 6 - - - - -

© 2016 Nature America, Inc. All rights reserved. IL-2 at 37 °C and 5% CO 5% and °C at 37 IL-2 transduced cells were cultured in the presence of 50 U/ml recombinant human incubation the transduction procedure was repeated. Prior to analyses, further cells were spinoculated at 600 5 of presence in added were LV-Brec1particles LV-Ctr of or amounts various pre-stimulation, Following ratio. 1:1 a at h 24 (Invitrogen) for beads magnetic anti-CD3/CD28 with equilibrated stimulated infection. after h 8 changed at 600 were spinoculated cells HIV-1 10 × (1 cells T PM1 transduction. after h 8 600 at spinoculated 5 of presence the at in cells amounts the to various added were particles RV or LV cells HeLa HIV-infected and of cultures. cell and Transduction infection described as were as particles determined fluorescent forming units split-packaging per exactly ml (ffu/ml) with cells 293T described as plasmids of expression cotransfection transient by produced detail in described previously backbone vector SIN alpharetroviral an into inserted LV-Ctr were LV-Brec1and from In RV-Brec1 and RV-Ctr, the respective 2TAR- the by deleting constructed was resulting in the promoter, construct SFFV LV-cBrec1.the by replaced A was negative lentiviralexpression) GFP control vector,(regulating promoter LV-Ctr, (EF1 alpha 1 factor elongation constitutive the by substituted was LV-Brec1 in element promoter 2TAR the analyses, toxicity EGFP. of various For expression constitutive directs promoter (PGK) kinase phosphoglycerate human the addition, In cells. infected HIV to expression of the target sequence HIV-1active Tat the under TAR (2TAR), placed the tandem repeat Brec1-resistant is of an control engineered Brec1 encoding sequence gene the (LV-Brec1), vector previously detail in described been has GFP and Brec1 of delivery for used (LV) backbone vector lentiviral replication-incompetent particles. viral of production and vectors Viral on Recombination above. described mg/ml 1 with induced was sion in tested was sites genomic human on Brec1 of (Stratagene) following the manufacturer’s instructions. Recombination activity pEVO-Brec1- of mutagenesis site-directed by generated were SeLOX sites genomic using respective The genome human the screening by identified were sites tion analysis. recombination Off-target ATCTT-3 F primers the using PCR by (5 recombination for assayed manufacturer’s using and the instructions following transfection (Qiagen) after Kit h Mini 48 Blood DNA QIAamp isolated the was DNA Genomic plasmid. expres sion recombinase of ng 400 with transfected and plates 6-well sites in grown target integrated stably with lines cell FACSDiva BD software. 8.0 using analyzed were Data sample. each of cates repli Technologies). FACS on technical three were performed measurements (Life excitation nm 640 for Kit Stain Cell Dead Red Far Fixable the LIVE/DEAD using stained were nm samples the 640 and discrimination 561 cell 488, live/dead For with lasers. equipped system II Canto Dickinson Becton a (FACS) sorting cell using by fluorescence-activated and was quantified EGFP mCherry of expression In addition, transfection. after h at 48 EVOSmicroscope FL the using imaging fluorescence by assayed were 1:4. cells of plasmid Co-transfected reporter fluorescent to plasmid expression recombinase of a of with ng total 400 of DNA and at plates transfected in a grown 6-well ratio nature biotechnology nature ′ For transduction of patient’s HIV-1 CD4 HIV-1 patient’s of transduction For were particles (RV) retroviral and (LV) lentiviral pseudotyped VSV-G HeLa reporter context, genomic the in activity recombination assay To -GCCTCGGCCTAGGAACAGT-3 BaL u2 n h peec o 1 of presence the in Luc2 ′ ) ( ) 2 Supplementary Fig. 3 Fig. Supplementary 9 loxBTR . g for 90 min at room temperature. Medium was changed changed was Medium temperature. room at min 90 for using the QuickChange Site-Directed Mutagenesis Kit Kit Mutagenesis Site-Directed QuickChange the using 2 . g g for 90 min at ambient temperature. After 24 h of for 90 min at room temperature. Medium was Medium for at 90 min temperature. room l 28 -arabinose and recombination assayed as as assayed recombination and -arabinose ). , 2 loxBTR µ 9 Potential off-target Brec1 recombina Brec1 off-target Potential . Likewise, titers of the respective viral viral respective the of titers Likewise, . ′ g/ml polybrene (Sigma-Aldrich) and and (Sigma-Aldrich) polybrene g/ml ad (5 R and ) brec1 trans brec1 µ 2 8 g/ml protamine sulfate. Cells were were Cells sulfate. protamine g/ml . served as positive control. positive as served -activator, thereby limiting -activator, Brec1 thereby limiting expression cassette in LV-Brec1.in cassette expression + 6 and/or For transduction of For PM1 T transduction cells T cells, cell cultures were pre- were cultures cell cells, T ) were infected with 500 ng of of ng 500 with infected were ) loxBTR µ E. coli E. The self-inactivating (SIN) (SIN) self-inactivating The g/ml protamine sulfate and and sulfate protamine g/ml α ′ -CCGCCACATATCCTG ) promoter, and the PGK PGK the and promoter, ) egfp , , . Recombinase expres Recombinase . loxP 2 expression cassettes cassettes expression 9 . In the respective respective the In . or or loxLTR were were cis 1 9 - - - - - .

according to the manufacturer’s instructions, except that a fourfold excess of excess fourfold a that except instructions, manufacturer’s the to according staining for surface used were antibodies #555700) BD Pharmingen, TRAP1; (clone CD154-PE anti-human mouse and #345771) Dickinson, Becton SK2; (clone CD4-APC anti-human mouse #641397), Biosciences, BD SK7; (clone described as previously performed tially essen was ICS instructions. manufacturer’s the to according controls, lated 10 × 1 from supernatants with performed was (Qiagen) ELISArray Multi-Analyte Cytokine Th17 Th2, Th1, Human (ICS). staining cytokine intracellular and Elispot ELISA, by monitored were levels (PMA) acetate and conc.) ionomycin (0.67 final (50 ng/ml myristate phorbol with h 12–24 for stimulated were cells T system. Dickinson) FACSCanto BD a (Becton using were harvested, stained according to the manufacturer’s protocol, and analyzed For 5 Dickinson). analysis, × 10 (Becton conjugate V-APC Annexin antibody the with together (Invitrogen) FACSCanto BD a system. using cytometry flow before h 2 for dark the in followed temperature ambient at was incubation min, further by 30 for °C 37 at Incubation Sigma-Aldrich). mg/ml; (0.5 33 and Sigma-Aldrich) mg/ml; (10 A RNAse and rehydrated in 450 were cells pelleted the for Afterwards, h 24 at °C. −20 incubated were cells the on for ice, min 20 EDTA and fixed drop-wise with 5 ml of 80% ice cold ethanol. After incubating 500 in suspended PBS, with washed harvested, were T Jurkat cells analyses. Safety vector, pCAGGS-IRES-puro, for constitutive expression of Brec1 from the the hybrid (CMV) immediate early beta-actin enhancer-chicken cytomegalovirus from Brec1 of expression constitutive for pCAGGS-IRES-puro, vector, mice. transgenic expression into mammalian the was subcloned NLS-Brec1 Codon-optimized Brec1-expressing of characterization and Generation used. array the on genes) RefSeq in kb (4.6 kb 5.3 was spacing probe overall median The 3. to set was aberration an nate desig to affected probes of number minimum The 2X400K. Kit Microarray CGH Human G3 SurePrint Agilent an on cells mock-transduced DNAfrom Blood Mini Kit (Qiagen) for array-CGH analysis. analyses. DNA was hybridized against QIAamp DNAthe using extracted DNA was (array-CGH) genomic and transduction after CD4 human hybridization primary LV-cBrec1-transduced genomic Array-comparative Imaging) coupled to an epifluorescence microscope (Leica). SKY probe mixture and analyzed using the SpectraCube system was(Applied dropped Spectralonto glass slides. Metaphase chromosomes were hybridizedat 37 °C for 15with min and fixedthe in 75% methanol/25% acetic acid. Cell suspension 0.1 mg/ml colcemid for 4 h. Cells were then treated with 75 mM KCl, incubated cellswerearrestedafterT transduction mitosisd in 7 treatingby cellswith the Spectral karyotyping (SKY) assay. CO counted. and 5% identified were colonies and °C 37 at incubation After dish. were culture cells cell diameter 3.5-cm a mock-treated into seeded and or methylcellulose of ml 1 in suspended transduced 100 Technologies). (STEMCELL (CFU) assay was methylcellulose using performed H4435-enriched methocult previously validated been have used Antibodies 3410-2A). Code: (Mabtech; IL4 anti-human antibodies detection biotinylated the using stimulated with PMA/ionomycin as indicated above. Secreted IL4 was detected 10 h. × 12 3 for °C Afterwards, 4 at saline phosphate-buffered in 3410-3-250) Code: Mabtech 50 with IgG1; ng (mouse of Millipore) IL4 antibody anti-human recombinant Technologies). (Life excitation nm 405 for Kit Stain Cell Dead Aqua Fixable LIVE/DEAD the using performed also was staining live/dead a staining, cytokine the to addition In used. was #560741) Pharmingen, BD mouse anti-human IFN staining, intracellular For stimulation. during cells the to directly added was antibody CD154 the For analysis of immune activation, LV-transduced primary human CD4 human primary LV-transduced activation, immune of analysis For V Kit FITC Annexin the using was performed events of apoptotic Analysis The differentiation potential of transduced HSC by colony forming unit unit forming colony by HSC transduced of potential differentiation The (96-well; plates polyvinylidene coating by performed was analysis Elispot To determine cell cycle distribution 1 × 10 × 1 distribution cycle cell To determine 3 to 1 × 10 × 1 to 5 5 cells were seeded on the coated plates and and plates coated the on seeded were cells LV-transduced human CD4 primary LV-cBrec1-transduced primary human CD4 2 µ 9 . Mouse anti-human CD3-APC H7 CD3-APC . Mouse anti-human l l of 16.6 with PBS supplemented µ M final conc.). Specific cytokine cytokine Specific conc.). M final γ + -PE -PE Cy7 (clone 4S.B3 antibody 6 µ cls ee avse 7 d 7 harvested were cells T stimulated cells or unstimu or cells stimulated l propidium iodide solution solution iodide propidium l 2 for 14 d, various cell cell various d, 14 for doi: 2 9 10.1038/nbt.3467 6 . LV-transduced LV-transduced µ l PBS/1% l PBS/1% + T cells µ + + - - - l

© 2016 Nature America, Inc. All rights reserved. performed for these and the following animal experiments. animal following the and for these performed version Roche Ltd.). and 2.0 Randomization (Hoffmann-La blinding were not Test HIV-1 TaqMan AmpliPrep/Cobas Cobas copies/ml) RNA HIV-1 (<20 mouse plasma with human serum (PAN Biotech GmbH) using the ultrasensitive using retro-orbital sampling. Likewise, viremia was assayed by diluting cell-free FACS analysis of peripheral blood samples at 4–21 weeks after transplantation, by verified was engraftment cell human of study. Analysis the from excluded CD4 into mice by i.p. injection. Animals that showed engraftment with >5% human transplanted were GmbH) (PANBiotech serum AB human 0.1% containing 10 × 1 Subsequently, Conservatome). (CSL-12; Gy/min 3.75 at source 137 cesium a animals were irradiated using a dose of 4 Gy (at 4 h before transplantation) from (FormuMaxClophosome-A-Clodronate Liposomes Inc.). 48 Scientific h later, were animals (i.p.) by of preconditioned injection 60–80 intra-peritoneal LV-Brec1)-transduced patient-derived CD4 Rag2 mice. humanized of analysis and Generation instructions. manufacturer’s the following RT (Invitrogen) III SuperScript manufacture’s the instructions. following (Qiagen) h. 2 for °C at 55 incubated was it DNA dissolve 100 in resuspended and dried DNA pellet the and removed was supernatant The min. 20 for °C 4 and speed maximum at by centrifugation pelleted it was min, for 30 °C at −80 DNA the precipitate 500 adding by DNA and precipitated tube new a to transferred was supernatant the min, 15 and 4 at speed °C for maximum in a microcentrifuge by debris centrifugation cellular other and hair pelleting After shaking. gentle at with °C 55 overnight 100 of concentration final a to K teinase 5 Tris-HCl mM 8.5), (pH EDTA mM 200 SDS, and NaCl) pro 0.2% 8.0), (pH synthesis. cDNA Genomic and DNAbiopsy was tail isolated mouse from from tail biopsyextraction RNA by and addingDNA 500 P1/P2). (primers activity enzymatic provide due to Brec1 product to excision and of circularized the for occurrence the P3/P4) evidence (primers cells mice the of genome the in sequences DNA kit) and was subjected to PCR analysis to analyze the presence of lentiviral 5% CO 650 at min 10 for culated 12.5 protaminewith sulfate (SIGMA)supplemented and were 125 IU/ml rhuIL2 cultures (Biomol). and Cells were 20 spinoc of (MOI) infection of ( reporter the carrying particles SIN lentiviral VSV-Gpseudo-typed later, h 6 well). per suspension plates, 96-well into well Dynabeads per cells 300,000 of seeding After PBS. with twice washed were cells and centrifugation by separated were PBMC instruction. manufacturer’s to according ice on min 15 within (SIGMA) buffer lysis cell (1,500 trifugation from or blood C57/Bl6 Brec1 mice were by was Plasma isolated. depleted cen 500 from PBMC tests, For recombination mR. and mF primers Brec1-specific the using detected was (cDNA) DNA RNA; complementary biopsy Brec1 tail from (RT-PCR) Brec1 PCR for transcription analyzed reverse and by above expression described transgene as the genotyping by carrying identified Offspring were (C57BL/6). mice wild-type with mated mR (5 (5 mF primers Brec1-specific the using genotyping PCR-based for extracted DNA genomic and obtained was ducts of pseudo-pregnant mice. After mouse pups were born, a small tail biopsy to the ovi transferred were surgically eggs fertilized the DNA, with surviving injection Following gel. electrophoresis a from preparative purified sequence was digested with HindIII and the 6.1-kb fragment containing the CAG-Brec1 construct the oocytes, (CAG) promoter. of fertilized For injection pronuclear doi: First-strand cDNA was synthesized by RT-PCR from mRNA using using mRNA from RT-PCR by synthesized was cDNA First-strand Total Mini Kit from RNAthe Protect using biopsy RNeasy tail was purified 10.1038/nbt.3467 + −/− cells were included, whereas animals displaying >20% weight loss were loss weight >20% displaying animals whereas included, were cells ′ 6 -TCCTTGATTCTGATCCGGGC-3 2 lentiviral vector (LV-Brec1 or LV-Ctr)-transduced cells in 150 150 in (LV-Brec1 cells vector LV-Ctr)-transduced or lentiviral , , 95% humid. Genomic DNA was isolated from the cells (Qiagen blood γ c −/− R Mouse T-Activator CD3/CD28 beads were added (30 were added beads Mouse T-Activator CD3/CD28 (hu-Rag2) mice engrafted with lentiviral vector (LV-Ctr or or (LV-Ctr vector lentiviral with engrafted mice (hu-Rag2) µ l isopropanol and inverting the tube ten times. After storing storing After times. ten tube the inverting and isopropanol l g , 2 min, 4 °C) and erythrocytes were lysed with red blood blood red with lysed were erythrocytes and °C) 4 min, 2 , Supplementary Fig. 6d Fig. Supplementary g . Afterwards, cells were incubated for 7 d at 37 °C, °C, 37 at d 7 for incubated were cells Afterwards, . ′ -AACAACCGGAAGTGGTTCCC-3 µ ′ g/ml to the sample and incubating incubating and sample the to g/ml + µ ). ). Founder mice were subsequently T cells, 6-week-old male or female l nuclease-free water. To the help nuclease-free l ) were added at a multiplicity multiplicity a at added were ) To generate “personalized” “personalized” generate To µ l lysis buffer (100 mM µ l peripheral peripheral l µ l original l original µ µ ′ l PBS PBS l ) and and ) g/ml g/ml µ l l of - - - -

of peripheral blood samples at week 16 after transplantation. Viremia was was Viremia transplantation. after 16 week at samples blood analysis FACS by peripheral of verified was Engraftment before. as Gy 1 with irradiated were newborns the transplantation), before h 4 at (i.e., injection i.h. to Prior CD34 transduced 10 × 3 with (i.h.) intra-hepatically NOD.Cg- newborn injecting by ated controls. staining tive nega as served transplants without mice from obtained cells and antibodies Isotype #25-0049-42). eBioscience, RPA-T4; (clone antigens CD4 and human (H130) the CD45 against directed antibodies monoclonal with staining by analyzed were mice Beckman HIV-1 infected Inc.). (eBioscience UCHT1; #A94680) Coulter, (clone CD3 human and #12-0459), eBioscience, H130; raised antibodies monoclonal with against mouse CD45.2 (clone stained 104; eBioscience, #47-0454), were human CD45 (clone cells collected r.o. ment, engraft cell human To monitor (Treestar). PC for v7/9 software FlowJo and BD FACSDivav5.0.3 with system Software a Dickinson) FACSCanto (Becton using paraformaldehyde 1% plus FACS-buffer in analyzed were cells Stained 70- sterile a through filtering and dissection tissue manual by necropsy at pared pre were liver and spleen of suspensions Single-cell antibodies. monoclonal with stained EDTA) and mM 2 and FCS 2% containing (PBS FACS-buffer in resuspended was pellet cell blood white The (Sigma-Aldrich). Buffer Lysing 10 mM EDTA) 100 and red blood into cells were (r.o.) lysed by treatment sinus with Red Blood Cell venous retro-orbital the 59. 58. 57. 56. 55. local Hamburg). the (Ärztekammer commission by ethics approved was which consent, informed written after obtained was material Patient 29/14). and 23/11 63/09, (Nr.: Verbraucherschutz und Gesundheit für Behörde Hamburg, Hansestadt und Freie the and PV4666) and WF-010/2011 OB-050/07, Hamburg; (Ärztekammer commission ethics local authority, the German relevant by the approved and reviewed were cols proto experimental The Law. Protection Animal German the of guidelines statement. Ethics previously validated been have used Antibodies Kit visualization. for used were Red Permanent TNB-Amplification with the and phosphatase alkaline at set Streptavidin cooker °C. pressure 100 a using boiled (Dako) were 1699 S buffer sections retrieval in Deparaffinized min 20 for dilution. 1:10 a in used was M0857) haemalumn. with terstained Kit (Dako) and diaminobenzidine were used as substrates. TNB-Amplification Sections were The coun visualization. for used was streptavidin peroxidase Biotinylated anti-mouse monoclonal antibody in combination with horseradish Monoclonal was in used a dilution. clone F7.2.38) M7254, anti-human CD3 1:1,000 (Dako detection. antigen CD3 human for overnight waterbath °C 85 an in buffer citrate in incubated were sections Deparaffinized analyzed. were analysis. Immunohistochemical previously validated been have used Antibodies before. described as TaqMananalysis by assayed Animals into which human CD34 human which into Animals 50 cells, of peripheral For analysis To visualize p24 antigen, the monoclonal antibody clone Kal-1 (Dako (Dako Kal-1 clone antibody monoclonal the antigen, p24 visualize To

µ Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M. & Barton, G.J. Jalview G.J. Barton, & M. Clamp, D.M.A., Martin, J.B., Procter, A.M., Waterhouse, K. Prüfer, H. Li, 2. Bowtie with alignment gapped-read Fast S.L. Salzberg, & B. Langmead, K. Anastassiadis, eso 2- mlil sqec ainet dtr n aayi workbench. analysis and editor alignment sequence Bioinformatics multiple 2--a Version Bioinformatics SAMtools. and format Alignment/Map Methods (2009). in recombinase m m nylon for mesh (BD and staining antibody Biosciences) FACS analysis. t al. et

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25 24 E. coli E. aMN rpd lgmn o sot eune t lre databases. large to sequences short of alignment rapid PatMaN: The animal experiments were performed according to the the to according performed were experiments animal The + et al. et cells in 30 30 in cells , 1189–1191 (2009). 1189–1191 , (2008). 1530–1531 , 2 2 9 9 . . , mammalian cells and mice. and cells mammalian , Dre recombinase, like Cre, is a highly efficient site-specific efficient highly a is Cre, like recombinase, Dre µ Formalin-fixed and sections wax-embedded l PBS containing 0.1% human AB serum. serum. AB human 0.1% containing PBS l 5 lentiviral vector (LV-Ctr or LV-Brec1)- or (LV-Ctr vector lentiviral µ Bioinformatics + l to 100 l to 100 HSC were transplanted were gener were transplanted were HSC Prkdc scid µ nature biotechnology nature µ IL2rg l of blood was collected from from collected was l of blood l bleeding-buffer (PBS plus plus (PBS bleeding-buffer l Dis. Model. Mech. Model. Dis.

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