© 2016 Nature America, Inc. All rights reserved. or or J.F.G. ( 12 Ewha Womans University, Seoul, Korea. Boston, USA. Massachusetts, Beijing, China. Sciences Abel Salazar, University of Porto, Porto, Portugal. General Hospital, Boston, USA. Massachusetts, Harvard Medical School, Boston, USA. Massachusetts, 1 to estimated rearrangements the been of abnormalities developmental relevance has the screening emphasizing con 1.5%, be of diagnostic to for 0.47% referred be samples to estimated newborns secutive was CNVs NAHR-mediated segment this of copy either in result (microduplication) gain copy can reciprocal or the and (microdeletion) loss segment genomic a flank are that SDs rearrangements homologous highly recurrent two between occurs which by NAHR, These mediated disorders. psychiatric and neurodevelopmental of human causes common most the are among (rMDS) syndromes microduplication and microdeletion Recurrent permit efficient correction of these defects. of genomic disorders and, with further development, may also This new approach will provide broad for applicability the study in vivo clusters signatures transcriptional from human subjects with The method is and reproducible, RNA sequencing reliably of one of copy-equivalent the SDs that mediate NAHR reciprocal CNVs of 16p11.2 and 15q13.3, including alteration disorders and demonstrate its capabilities by generating repetitive elements (SCORE), to model reciprocal genomic engineering method, single-guide CRISPR/Cas targeting of modeling tool. We describe here a CRISPR/Cas9 genome variants (CNVs) that mimic NAHR would represent a valuable capability to efficiently generate reciprocal copy number a challenge for modeling these syndromes; however, the syndromes. The genomic architecture of flanking SDs presents of human disease, often producing distinct phenotypically identical segmental duplications (SDs) are a major cause non-allelic homologous (NAHR) recombination between near- Recurrent, reciprocal genomic disorders resulting from Mustafa Sahin Catarina M Seabra Derek J C Tai CRISPR with duplications segmental targeting by microduplications and microdeletions Engineering nature nature Received 8 May 2015; accepted 7 January 2016; published online 1 February 2016; Molecular Neurogenetics Molecular Unit, Neurogenetics Center for Human Genetic Research, General Massachusetts Hospital, Boston, USA. Massachusetts, Department Department of Genetics, Harvard Medical School, Boston, USA. Massachusetts, should Correspondence be addressed to M.E.T. ( CNVs and their corresponding [email protected] NEUR 7 Department Department of Laboratory Medicine, Boston Children’s Hospital, Boston, USA. Massachusetts, OSCI 1 1 – . In one study, the prevalence of five common common five of prevalence the study, one In . 8 4 , , Chengsheng Zhang EN 2 , , Ashok Ragavendran , and the rate of recurrent rMDS in prenatal prenatal in rMDS recurrent of rate the and , C 1 E –

9 5 The The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA. advance online publication online advance , Serkan , Erdin Serkan 3 . 11 ). Program Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, USA. Massachusetts, in in vitro 4 Department Department of Psychiatry, General Massachusetts Hospital, Boston, USA. Massachusetts, 9 1 , Charles , Lee Charles , 3 1 3 , Psychiatric Psychiatric and Genetics Unit, Neurodevelopmental Center for Human Genetic Research, Massachusetts , 4 models. 3 , , Ryan L Collins , 4 6 Beijing Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, , , Poornima Manavalan

in in vivo

9 , 10 , , James F Gusella .

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1 , 3 , including obesity traits, including anthropometric as well as disorders, neuropsychiatric other and schizophrenia disorder, spectrum autism disability, intellectual with associated been has that rMDS common a is 611913) (OMIM ases (TALENs) ases (ZFNs) nucleases tool engineering genome applicable broadly and immunity adaptive complex involved in bacterial interfering ered as a ribonucleoprotein discov was complex (Cas) (CRISPR)/CRISPR-associated neurodevelopment. human in rMDS of impact the understanding to approaches innovative to access open would cells, neural derivative and precursors neural ing includ tissues, relevant into differentiated be then can that (iPSCs) methods to model rMDS alterations humans in induced pluripotent stem to cells generalizability their in limited be may models animal Meanwhile, cohorts. large collect to inability the and tissues peripheral available of relevance uncertain the background, genetic by factors in several studies of humans confounded and their including cells, variable are it in individual of and CNV entire the of SD the of copy-equivalent one and ment seg genic of unique the gain of or copy one loss encompassing the segment either 740-kb a in result can which SDs, flanking the of formation CNV NAHR-mediated coding as annotated (5 genes cated (ref. kb 147 spanning each SDs, identity) V.71 (ref. GRCh37 Ensembl on (based coding protein as annotated are (ref. kb 593 spans CNV 16p11.2 of the segment genic SD. of unique the The copy-equivalent one and segment genic unique a of loss or gain involves rMDS 16p11.2 ders, 4 doi:10.1038/nn.423 , , Ian Blumenthal Reciprocal CNV of a small segment of 16p11.2 16p11.2 chromosome of segment small a of CNV Reciprocal The clustered regularly interspaced short palindromic repeats repeats palindromic short interspaced regularly clustered The 6 )). It is flanked by parallel and highly homologous (>99% (>99% homologous highly and parallel by flanked is It )). 1 , 3 , 4 , , Alexei Stortchevoi 1 1 , 3 11 , CRISPR/Cas has inherent endonuclease activity activity endonuclease inherent has CRISPR/Cas , 1 4 5 , 2 . Like all such NAHR-mediated genomic disor genomic NAHR-mediated such all Like . 12 1 and transcription activator-like effector nucle effector activator-like transcription and 0 10 , and it has now evolved into a programmable programmable a into evolved now has it and , & Michael E Talkowski 8 1 Department Department of Neurology, Boston Children’s Hospital, Department Department of Graduate Studies – Life Sciences, , Xiaoli , Chen Xiaoli e t c 5 n h ) containing 47 genes, of which 28 28 which of genes, 47 containing ) n vivo in 1 , 3 [email protected] , 4 6 C I 2 , , , Yiping Shen 7 Department Department of Neurology, 5

. Disentangling the effects effects the Disentangling . involves the mispairing mispairing the involves ) and containing 6 dupli 6 containing and ) p e r l a 5 6 Institute Institute of Biomedical ). The mechanism of of mechanism The ). 1 1 1 . Like zinc finger finger zinc Like . – 4 , 11 7 o , 8 ,

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© 2016 Nature America, Inc. All rights reserved. ing confirmed the breakpoint precise NHEJ and ing as confirmed revealed the likely ( 29,624,445–30,199,859) 16: chromosome V.71) GRCh37 (Ensembl intact fully SDs flanking the leaving while microdeletion 575,414-bp predicted the generated experiment This Methods). Online (see SDs the between segment genic contiguous mapping the and dgRNAs within uniquely targeted two independent ( group comparison initial an as segment genic in unique the genes all containing contiguous sequence 575-kb a of deletions heterozygous create to approach proven strategy dgRNA a using inversions and tions that the system CRISPR/Cas can generate dele efficiently predictable shown previously been ItSDB. as has ) the proximal to (closest SD the and SDA as telomere) the to (closest SD distal the to Methods; Online (See trols con family-based as well as microduplication) with subjects three microdeletion, with subjects (four screening previously clinical were from that detected rMDS 16p11.2 harboring subjects gener from lines ated iPSC used we controls, positive As sample. test the as All studies used a previously described control iPSC line (8330) line iPSC control described previously a used studies All rMDS. An overview 16p11.2 of our common experimental design is the shown in on hypothesis this tested initially We rMDS. NAHR-mediated recurrent in those to comparable directly CNV cal recipro generate to iPSCs human to applied be could technologies engineering genome emerging in optimizations that Wepostulated Generation of reciprocal 16p11.2 CNVs using CRISPR/Cas9 RESULTS anomalies. developmental common of causes these to human access modeling open widespread with SDs, the can SCORE of that suggest data These efficiency. of rates copy-equivalent reproducible one of duplication or deletion including mechanism, NAHR the of products the mimic that CNVs and 16p11.2 In 15q13.3. each region, the generates method reciprocal composition, and size differing of regions rMDS proof-of-principle method to rapidly and generate efficiently reciprocal CNVs using two homologous sequences in the SDs. We demonstrate the capability of RNAthe target perfectly to a (sgRNA) directly uses strategy single-guide ToSCORE SDs. this, flanking accomplish of NAHR between quence conse a as humans in disorders genomic reciprocal with associated producing microdeletions and microduplications comparable to those rearrangements genomic other or deletions produce to strategy (dgRNA) RNA dual-guide a in used be can gRNAs of pairs recombination homologous by repair permit to cell into the a can be delivered mutation a carrying donor template Cas9, point precise mutations at or insertions a target target gene cleaved by the of expression normal abolish that mutations indel induce can (NHEJ) end-joining non-homologous gRNA-directed Cas9 nuclease-induced double-strand breaks through to generate point mutations or of deletions a genic ofRepair segment. in cells editing genome is targeted system crRNA to complementary is that sequence target specified a to Cas9 direct fixed a and crRNA a of chimera a as RNA, or gRNA) RNA guide (a engineered a single laboratory, In the and RNA (crRNA) CRISPR with complex II type system the in protein CRISPR/Cas Cas single a on rely CRISPR/Cas of cations (PAM) motif 5 proto-spacer-associated sequence, a with along target encoun its it ters when break double-strand a DNA in generate can that e t 

In this study, we present a method, SCORE, for efficiently efficiently for SCORE, method, a present we study, this In c 1 n h 4 . . One of the most for common applications the CRISPR/Cas ′ -NGG, where N is any where nucleotide -NGG, C I p e r l a 1 5 Cs, hc i is ot atra om a forms bacteria host its in which Cas9, , Supplementary Fig. 1 Fig. Supplementary Fig. 1 Fig. o t r b ). This initial experiment used used experiment initial This ). trans s 1 6 trans Fig. 1 Fig. and model organisms and model -activating crRNA can can crRNA -activating 1 4 1 -activating crRNA -activating . Most current appli . Most current 8 . We first used this this used first We . b ). Sanger sequenc Sanger ). ). We refer herein herein We ). refer 1 6 1 . To introduce introduce To . 1 . In addition, addition, In . 16 , 18– Figure 1 2 1 . 8 1 , 2 1 a 4 2 7 ------. .

Once clonal iPSC colonies were formed, relevant gene dosage was was dosage gene relevant formed, were colonies iPSC clonal Once plates. isolate 96-well to Matrigel-coated of (FACS) wells individual into iPSCs sorting single cell fluorescence-activated by followed Methods, in Online described methods transfection sgRNA CRISPR the by copy-equivalent produced one is as and SDs, the of segment comprising genic unique 593-kb the 29,487,574–30,226,919) of all 16: (chromosome deletion We promote would of formation a strategy this 739,346-bp predicted the in elsewhere not ( but genome SDs, both in breaks DNA simultaneous promote to SDB and SDA of each in site homologous a We targeted ( lesions rMDS to model SCORE, method, this of single-guide new potential the Weevaluated humans. in CNV the of size the mirroring SDs, in of gene the each and one copy-equivalent segment genic unique the encompassing one that is, that rMDS NAHR; by 16p11.2 mediated is the of microdeletion 740-kb a generate could SD flanking each in sites homologous perfectly targets that sgRNA vivo in SDs. the of copy-equivalent one and segment unique this alters NAHR where humans, in rMDS reciprocal model accurately not does segment genic unique the deleting for strategy ( mechanism repair nucleotide T insertion was found in four deletion lines (Del 5–8), 5–8), (Del lines deletion single- four in a found 4), was (Del insertion T line nucleotide deletion one in found were mutations 1–3), two (Del lines microdeletion three in found were mutation indel ( lines plication microdu 5 and lines microdeletion 740-kb CRISPR-treated 12 the and Sanger sequencing of the region. We found clear junctions among cloning by sgRNA sites alterations for targeting performing potential sites. predicted most the explore to at able were we sequence experiments, our in However, SDB and SDA the the to owing distinguish to accomplished inability been not has subjects rMDS of 16p11.2 SDs in points exchange precise the of identification our To knowledge, SDs. the within CRISPR/Cas by generated breakpoints the sequence and clone to attempted we iPSCs, SCORE-generated with in this segment may be incompatible with iPSC viability in our system. 16p11.2 rMDS region, suggesting that total absence of one ( or more genes Notably, no off-target CNVs were detected from genome-wide( microduplications analysesand microdeletions observed the ( DNAmicroarray analysisscreen fortoon-target andoff-target CNVs genome-wideandtranscriptomelines) total sequencing(RNAseq; 23 of select dosage-altered clones were then performed by strand-specific respectively,analysesFurther5,clonesscreened). and 114 (12of tion putativemicroduplicaharboreda clones of 4.3%microdeletion, and expected 740-kb deletion, suggesting an overall efficiency of 10.5% for analysis( repeatedand 114 clones were systematically screened by copy number approach,entireexperimentwas thethe ofTo efficiency estimatethe iPSCs CNV 16p11.2 of characterization and Replication the mimic NAHR. of that consequences imbalances dosage reciprocal predictable promote can method SCORE our that suggesting microduplication, apparent with consistent dosage 16p11.2 increased with clones identi fied also assay The microdeletion. expected the with consistent dosage decreased showed that clones CRISPR/Cas-modified tified iden readily screening This screening. number copy by measured Supplementary Fig. 4 Supplementary Fig. 3 Fig.Supplementary To create a method that would mimic the consequences of the the of consequences the mimic would that method a create To To provide insight into the rearrangement mechanism associated associated mechanism rearrangement the into insight Toprovide AR ehns, e yohszd ht rnfcig a transfecting that hypothesized we mechanism, NAHR Fig.1 Fig. 1 Fig. advance online publication online advance c b ). Inreplicatethis). experiment, clones12 harbored the Supplementary Fig. 5a Fig. Supplementary ; see Online Methods for details of sgRNA design). design). sgRNA of details for Methods Online see ; Supplementary Fig. 2 Fig. Supplementary ). We did not obtain homozygous deletions of the ). Dosage microarray and RNAseq confirmedRNAseq andmicroarray Dosage ). in vivo in , b mechanism. We used the the Weused mechanism.

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© 2016 Nature America, Inc. All rights reserved. ( while control lines clustered together with the control family member the corresponding patient deletion and duplication lines, respectively, all that revealed region CRISPR-treated rMDS deletion and duplication lines clustered 16p11.2 together with the of data the from transcriptome clustering Hierarchical line. microdeletion 575-kb the of CNV rMDS RNAseq from SDs the in genes 16p11.2 of reduction no the was there expected, harboring families from lines cell lymphoblastoid in previously described patterns transcriptional the four ( from copies) five to (increase lines microduplication the in SDs the of copy extra of one gain the and ) homologous two the across SDs the of copies total three to four from (reduction 740-kb lines the deletion from SDs the to localized genes the of copy one of loss respectively ( lines, microduplication SCORE- and 740-kb the microdeletion for segment generated genic unique the in genes all of overexpression and expression reduced revealed analyses RNAseq ( carriers CNV 16p11.2 of controls member CRISPR treatment that did not produce a CNV and those from family tion 740-kb microdele either CRISPR-treated with lines patient 16p11.2 lines, lines, microduplication microdeletion 740-kb and 575-kb NAHR. including potentially mechanisms, ogy-mediated microhomology-mediated mechanisms but likely also involve homol tion and microduplication lines, which do involve nonhomologous or of microdele for CRISPR-treated repair mechanisms these disparate ( lines single-nucleotide duplication a micro 740-kb CRISPR-treated five of four in found was insertion T only Intriguingly, ( 9~12) (Del homology-mediated repair potential suggesting breakpoints, the at mutations new of dence and four deletion junctions contained no evi observed in CRISPR-generated clones. normalized log or losses of 16p11.2 region were determined by 16p11.2 region in CRISPR-treated lines. Gains Del) and duplication (CRISPR Dup) of the ( (5). clones microduplication all for and (6) clones microdeletion of a subset for performed was RNAseq and microarray by characterization Further genes. six for assay screening number copy by determined as SDs, the targets that method SCORE single-guide the using rMDS 740-kb the of generation CRISPR/Cas9 of ( 30,226,917–30,226,935). and 29,487,572–29,487,590 16: chromosome 5 sequence (target CNV NAHR-mediated of a model promote to SDs the targeting RNA single-guide the red deletion, 575-kb dual-guide the for segment genomic annotation V.71 GRCh37 Ensembl shown. are genes coding protein only simplicity, For SDs. and/or segment ( hybridization. genomic comparative array aCGH, procedures. experimental the of design ( CRISPR/Cas9. by iPSC human 1 Figure nature nature protein of levels altered confirmed also segment, genic unique the d b Fig. 2 Fig. Fig. 2 Fig. ) Microarray analyses showing deletion (CRISPR ) Illustration of targeted 16p11.2 rMDS rMDS 16p11.2 targeted of ) Illustration RNAseq was performed for 23 lines, including CRISPR-treated CRISPR-treated including lines, 23 for performed was RNAseq 2 3 or and microduplication, two of types control from lines: those b a

). Moreover, we observed expression patterns reflecting the the reflecting patterns expression observed we Moreover, ). ). In addition, western blot analysis of MAPK3, encoded in in encoded MAPK3, of analysis blot western addition, In ). NEUR Generation of 16p11.2 rMDS in rMDS 16p11.2 of Generation 6 . Yellow shows the targeted unique unique targeted the . shows Yellow 2 ratios. No off-target CNVs were OSCI Fig. 2 Fig. ′ -ACATGCCTATATCGCATAG-3 Supplementary Fig. 5a Fig. Supplementary EN upeetr Fg 5b Fig. Supplementary a C ). These These ). E

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QPR The unique genic segment contains seven protein encoding encoding protein seven and, when combined with one contains copy-equivalent 30,910,306–32,445,406) of the 15: flanking SVs, the chromosome segment GRCh37 (Ensembl genes genic unique The ity and variable facial and digital dysmorphisms (OMIM 612001) ciated with mental retardation, epilepsy, autism, schizophrenia, obes flanking SDs in 15q13.3. Recurrent rMDS of 218-kb this and regionsegment genic haveunique been 1.771-Mb a asso encompassing region rMDS larger, much yet common, another targeting study, second a To demonstrate generalizability of the SCORE method, we performed rMDS 15q13.3 the in approach SCORE the of Replication the harboring models yield comparable expression profiles to those of human iPSCs models mouse and humans in the 16p11.2 rMDS region ( Genome-wide differential expression was most significant for genes in upregulated, and respectively, downregulated relativesignificantly were coding) to protein controls (20 genes 25 (nominal deletion and microduplication lines, 28 genes (22 protein coding) and a linear increase in expression with copy number. In the 740-kb micro 32 genes detected by RNAseq (25 protein coding) in the region showed cal results in the CRISPR-treated iPSCs to those previous all detected; CNV and in the cortex of mouse models 16p11.2 harboring = state 1, 2 or in as study families 3), in a previous example, a linear trend in (for gene expressionstate copy must be with observed with copy correlated negatively or positively be to dosage gene level. We the also assessed at results from a controls linear model to requiring gene duplication) or (deletion genotype each compare CNVs 16p11.2 ( with lines iPSC CRISPR-treated our in expression Supplementary Fig. 6 Fig. Supplementary T ( n We applied statistical analyses based on general linear models to to models linear general on based analyses statistical Weapplied by FACS Single iPSCisolation = 12) ( n 4.3% = 114) Dup 29.8 ( KIF2 n MAZ PRRT = 5) 575-kb CRISPR/Casdeletion 740-kb CRISPR/Casdeletion PAGR PAGR1. 2 2 in in vivo MVP 1 d CDIPT 29.9 log2 ratio log2 ratio 2 +1 +1 –1 –1 0 0 SEZ6L2 16p11.2 CNV. 16p11.2 ASPHD1 CRISPR Del CRISPR Dup aCGH analysis Copy numberand KCTD13 ). Fig. 2 e t TMEM219 30. TAOK 0 HIRIP3 INO80E 5 c DOC2A , c 9 ), again replicating previous findings , FAM57B 2 2 n h 4 . These results imply that that imply results These . ALDO 30. RNAseq PPP4C 9 C I . . We largely observed identi 1 TBX YPEL A GDPD3 6 MAPK3 p e r l a 3 30. CORO1A 2 P genomic analysis Integrative functional BOLA < 0.05) ( SLX1 SULT1A RP11-347C12. 30.25 RP11-347C12. SDB A 2 o 3 Fig. 2 in vitro in (Mb) t r 25– 3 1 a 2 s 7 ).  - - - - .

© 2016 Nature America, Inc. All rights reserved. transfection approach as in the 16p11.2 CNV, followed by single-cell CNV, 16p11.2 the by in single-cell as approach followed transfection these flanking SDs (see (ref. total genomic imbalance due to NAHR in affected subjects is 1.989 Mb studies previous in reported as region, rMDS 16p11.2 the in genes the from signal strongest the see we red; in highlighted is 16 CNV. the of Chromosome function a linear as expression of model linear from ( lines. iPSC patient and lines treated CRISPR/Cas9 all for linkage, average using clustering, hierarchical difference mean back-transformed the represent bars error model; linear generalized the from contrasts on based controls all and lines CNV the between gene each for expression of difference mean the as calculated were changes Fold circles). blue (filled lines microduplication 740-kb CRISPR/Cas9 and circles), red (filled lines microdeletion 740-Kb CRISPR/Cas9 circles), red (open line deletion 575-kb CRISPR/Cas9 selected the for shown is regions SD and 2 Figure e t  c a

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(Mb) AC009133.14 Dup n PRRT2 = 2) PAGR1 PAGR1.2 MVP CDIPT SEZ6L2 SCORE strategy that targets the SDs. microdeletion and microduplication using the of CRISPR/Cas9 generation of this 2-Mb triplicate. Data are mean ± s.e.m. ( (deletion and duplication). Experiments are in eleven iPSC lines harboring 15q13.3 rMDS screening of all genes in the segment identified 32,799,503–32,799,521). ( : 30,792,593–30,792,611 and sequence: 5 of NAHR-mediated CNV is indicated in red (target The sgRNA targeting the SDs to promote a model protein-coding genes shown (Ensembl GRCh37). 15q13.3 rMDS segment and flanking SDs, with all iPSC by CRISPR/Cas9. ( Figure 3 Notably, these relatively high efficiencies for efficiencies high relatively Notably,these screened; clones 164 of (2 plication microdu for 1.2% and screened) clones 164 ~2 Mb CNV was 5.5% for microdeletion (9 of ofciency the SCORE approach for this larger, ( region CRISPR-targeted the flanking genes tion, and no dosage changes were detected for duplica single a beyond increase dosage no deletion of the 15p13.3 segment. We observed that were consistent with homozygous micro ASPHD1 upeetr Fg 7 Fig. Supplementary c KCTD13 ) Genome wide wide ) Genome

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© 2016 Nature America, Inc. All rights reserved. modestly to 5.5% when the deletion size increased to the 2 Mb of of Mb 2 the to increased size deletion the when 5.5% to modestly with a cut site single the was only reduced microdeletion 16p11.2 740-kb generating of efficiency SCORE 10.5% the that found we trast, than 100 kb greater for (ref. deletions than 1% efficiency less has NHEJ upon reliant is that CRISPR dual-guide that suggest studies Previous generated. initially CNVs 16p11.2 740-kb smaller much the of that to comparable is that efficiency an with approach SCORE the with derived be can models CNV reciprocal large tively efficiencies. relative their and repair of mechanisms these varied apart tease to needed expected be will be experiments would Further events rare. be to such though screening, dosage our by detected be not would that state dosage a balanced to leading other, possible on the duplication and also on one chromosome is may occur It deletion that possibility. this exclude cannot we though cells, of fraction meaningful a in mosaicism suggest not did results array micro and RNAseq the of Scrutiny detect. to challenging be would This population. cell expansion, final the in clonal mosaicism of levels low to during leading occur could exchange chromatid sister Nonetheless, expansion. by clonal followed cells, single ing to obtain FACS sort we performed mosaicism, for potential the To minimize factors. other and breakpoint mosaicism by multiple influenced of be could presence compositions the and method. efficiency this this using Notably, generated microdeletions of fraction a of and microduplications of including production the in possibly important be repair, may NAHR, homology-mediated that suggest data insertion ( single breakpoint at the a of a T or nucleotide microdeletions some for sequence wild-type as well as microdeletions, and the for NHEJ repair both of microhomology-mediated evidence observed we lines, microduplication SCORE-generated the Among system. this in operate also anisms mech non-NAHR that suggest recovered in fact The sequences junction numbers. equal roughly in recovered be which to expected duplication, be or might deletion either with cells individual to lead then would FACS before sorting mitosis in events of the segregation chromatids, sister affected alterations NAHR-mediated if However, in a cell would single not in be our for detected screen dosage. altered events duplication and deletion coincident on produced NAHR that microduplications and ( SCORE using microdeletions of generation the in nate predomi may mechanisms molecular different that suggest CNVs genic unique the region. any rMDS in SDs the and interrogation segment including anomalies, congenital of human causes common of these modeling genome-wide to access opens thereby SCORE SDs. flanking the of copy-equivalent one of imbalance dosage with reciprocal relatively including efficiency, high rMDS NAHR-mediated of outcome the model to possible is it that sizes of range a across segments genic unique within repair non-homologous by mediated variation structural of classes multiple generate can editing genome vivo. in produced those mimic that lesions generating by disorders genomic ing can emulate the genetic architecture of NAHR-mediated recurrent We show here that carefully designed CRISPR/Cas9 genome engineer DISCUSSION efficiencies. higher still in result could regions other or this in development Further optimization. without experiment initial single a on based detected were CNVs large such nature nature decrease efficiency corresponding the microduplication, For 15q13. The generation of 15q13.3 rMDS CNV also confirmed that rela that confirmed also CNV rMDS 15q13.3 of generation The CRISPR/Cas-generated the in observed sequences junction The Previous studies have shown that dual-guide CRISPR/Cas9 CRISPR/Cas9 dual-guide that shown have studies Previous NEUR OSCI Supplementary Fig. 5 Fig. Supplementary EN C 18– E

20 advance online publication online advance , 2 9 . This SCORE approach demonstrates demonstrates approach SCORE This . Supplementary Fig. 5b Fig. Supplementary ). A mechanism based purely purely based mechanism A ). 1 9

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of rMDS in multiple tissue types and, with further development and and development further with and, types tissue multiple in rMDS of modeling the enable will on which method, this details of application and complete design provide We models. mouse and families the of studies transcriptome and screening, genome-wide following CNVs off-target observe not did we as fidelity, high with models generates of suggest that the analyses rMDS the regions. Genome-wide method rMDS produce CNVs of different size and to copy state; we 0, 1, observed 2 implemented and 3 copies rapidly be can SCORE that reveal experiments Our anomalies. congenital human of causes common most the of many drives which NAHR, of consequences the models thereby derives recurrent, reciprocal dosage imbalances. This method and segments genomic unique SDs flanking homologous highly gets dence in humans ders (1 commonly observed in for 40,000 inci population the that note CNV is 15q13.3 one of disor genomic reciprocal the larger we recurrent but determined, be to remains approach SCORE the of limitations size precise The alone. repair non-homologous on utes to a of greater efficiency CNV formation than in methods relying contrib repair of homology-mediated that indicate the participation It may a across also distribution. broad size be tractable of will rMDS was from 4.3% to 1.2%. This suggests that relatively efficient modeling 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial studies. D.J.C.T., J.F.G. and M.E.T. wrote the manuscript. 16p11.2 family tissue samples. C.Z. and C.L. anddesigned performed microarray performed computational and statistical analyses. X.C., Y.S. and M.S. obtained P.M., C.M.S. and A.S. performed molecular studies. andR.L.C. A.R., S.E. M.E.T., J.F.G., D.J.C.T., A.S. and I.B. conceived and the designed studies. D.J.C.T., Taiwan (102-2917-I-564-012). Research Abroad Program sponsored by of Ministry andScience Technology of University Distinguished Professorship. D.J.C.T. is a recipient of the Postdoctoral of Mental Health to C.M. Lajonchere. C.L. is supported by an Ewha Womans is supported, in part, by grant from1U24MH081810 the US National Institute and the participating AGRE families. AGRE is a program of Autism Speaks and (C.L.). We gratefully acknowledge the resources provided by the AGRE consortium (C.L.) and National Research Institute award U41HG007497 supported also partially by US National Cancer Institute award P30CA034196 (M.E.T.) and Autism Speaks (J.F.G.). Research reported in this publication was (J.F.G.P01GM061354 and M.E.T.)), the March of Dimes (M.E.T.), NARSAD Institutes of Health (J.F.G.(R01NS093200 and M.E.T.), (M.E.T.),R00MH095867 the Nancy Lurie Marks Family Foundation (J.F.G. and M.E.T.), the US National Autism Research (SFARI 328656 and 346042 (M.E.T.) and SFARI 308955 (J.F.G.)), line. These studies were supported by funding from the Simons Foundation for Massachusetts Hospital)General for generously providing the control iPSC We thank S. Haggarty and S. Sheridan (Center for Human Research, Genetic online version of the pape Note: Any Supplementary Information and Source Data files are available in the the of in version available are references associated any and Methods M imbalances. genomic common these route to a provide could tractable optimization, COM AU Acknowledgment

ethods In conclusion, we describe here a method using a sgRNA that tar that sgRNA a using method a here describe we conclusion, In T Stankiewicz, P. & Lupski, J.R. Genome architecture, rearrangements and genomic and rearrangements architecture, Genome J.R. Lupski, P.& Stankiewicz, disorders. H P O 2 ET in vitro in 5 R R , 0.48% of cases of neurodevelopmental abnormalities neurodevelopmental of cases of 0.48% , I CONT NG the pape the TrendsGenet. FI edited lines recapitulated previous findings in rMDS rMDS in findings previous recapitulated lines edited N l RIBU . A NC r . T r IA . s I

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© 2016 Nature America, Inc. All rights reserved. medium for 3 d after Nucleofection. sequent screenings. For cell sorting, the iPSC were recovered in fresh Essential 8 intoMatrigel-coated 96-wellplates individually (one colony well),for per sub with puromycin (0.1 tion, iPSCs were harvested 24 h after nucleofection in fresh Essential 8 medium Replacement (KOSR) medium with feeder cellsSerum overnight.KnockOut iPS Forhuman incubating puromycinby made selec was which Biotech), Cruz supplementedand10 (R&D)bFGF ng/ml 10 with (CM) medium conditioned MEF using MEF) fibroblasts; embryonic (mouse inhibitor (Santa Cruz Biotech), or (ii) co-cultured on a monolayer of feeder cells wellsusing Essential 8 medium (Invitrogen) supplemented with 10 Matrigel-coatedon (i) settings:different two in cultured were iPSCs the tion, Cell programs Stem B-016, according to the manufacturer’s Human instructions. After nucleofec with used (Lonza) device II Nucleofection Amaxa Transfectionand (Lonza) 1 KitNucleofector vectors. pGuide-sgRNA or vector 5 (sg569: RNA guide chosen 2 For the SCORE method, the Human iPSCs (5 × 10 ( productjunction ~1-kb forexpected GFP an by of presence PCR the by of screened isolated level were colonies high were iPSC from a DNAs Genomic cells for expression. gated single (FACS) sorting and cell dissociated fluorescence-activated were iPSCs transfection, after h 72 colo Atdetails). forMethods Online iPSC (see puromycinselection after nies individual collected and RNAs guide both carry which Vectors pSpCas9(BB)-2A-Purowithhuman iPSCsfected gRNA_Cloningandplasmid a humidified atmosphere with 5% CO dish (Corning) with Essential 8 medium (Invitrogen) and incubated at 37 °C in Matrigel-coated on maintained were fibroblasts, adult from derived (iPSCs), C manufacturer’s instruction (Qiagen). the to accordingKit Maxi Plasmid EndoFree from purified were plasmids all gRNAthe in vectorconfirmed bywasSanger Sequencing.Before transfection, plasmid 41824) using a BbsI restriction site tion site restric BbsI a using 48139) plasmid Addgene (pX459, marker resistance cin 593–30,792,611 and 32,799,503–32,799,521 the following single-guide RNA was used: 572–29,487,590 and 30,226,917–30,226,935 the following single-guide RNA was used: arising from updates to the reference genome. and regions with the new assembly (GRCh38) to ensure there were no artifacts mitigating any possible off-target effects. Finally, we cross-verified the target didatefourthanlesswithguidesmismatches sitesacross nontarget regions, further methodology by using a recently released tool, Off-Spotter effects. Additionally, for the 15q13.3 guides, we refined our originaloff-targetpredicted respectively,no guideSDs, with 15q13.3 design and 16p11.2 the target performed a degenerate BLAST search to identify sequences thatJellyfish with wouldguides 18–25mer possible uniquely all identified first approach,we 30,199,842–30,199,861 29,624,428–29,624,447 the segment, rMDS the in genes sequences of unique dual-guide RNAs were as follows: all includes that microdeletion ence sequence arerefer based on genomeand assemblyRNAs GRCh37. To guide generateof the 575-kb design The segment. rMDS 16p11.2 the targeting tools.geno G ONLINE METHODS doi: ell culture and culture ell uide R µ All gRNAs were cloned into pSpCas9(BB)-2A-Puro plasmid with a puromy 5 sg387: For generation of the 2-Mb microdeletion and microduplication in 15q13.3, 5 sg569: For generation of the 740-kb microdeletion and microduplication in 16p11.2, To design the sgRNAs targeting 16p11.2 SDs and 15q13.3 SDs with the SCORE g total DNA plasmid, Cas9-sgRNA expression vector (pX459) with the the with (pX459) vector expression Cas9-sgRNA plasmid, DNA total g 10.1038/nn.4235 RA4: 5 gRNA841: 5 gRNA427: 3 N 1 . gRNA841 was cloned into gRNA_Cloning Vector (pGuide, Addgene A design and preparation. me-engineering.org ′ ′ -GCCTTAGGGGATTGCGGGAC-3 -GACATGCCTATATCGCATAG-3 DN ′ ′ -GCCTGGACACCGGGCGCAGG-3 -GCAGTGGCAGGCCATGAGCT-3 µ A transfection. A g/mL). After 24 h, surviving colonies were then collected ′ -GACATGCCTATATCGCATAG-3 / ) 3 1 o ban h sqecs f ud RNAs guide of sequences the obtain to We used the CRISPR Design Tool ( 2 Human induced pluripotent stem cells cells stem pluripotent induced Human . For the dual-guide approach, we trans 3 4 . Validation of the guide sequence ′ crmsm 1: 29,487, 16: chromosome , ′ 5 crmsm 1: 30,792, 15: chromosome , µ cells) were transfected with M ROCK inhibitor ROCK M (Santa Supplementary Fig. 2 Fig. Supplementary ′ ′ , : 16: chromosome , 3 , chromosome 16: 16: chromosome , 3 , to screen out can ′ ) and EGFP EGFP and ) µ M ROCK MROCK http:// 3 2 and ). ------

Foundation Autism Research Initiative. instruction. Genomic DNA for 15q13.3 patients was obtained from the Simons extraction by DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’sperformed we lines, iPSC patient 16p11.2 from extractionDNA genomicFor min. temperature10room forat incubation A RNasefollowedby min, 10 for were digested at 55 °C for 30 min followed by Proteinase K inactivation at 95 °C by adding DNA extraction buffer containing Proteinase K (0.2 mg/ml). Samples sionvector (pX459)usingRapid a DNA extractionmethod the genomic DNA was isolated from cells transfected by the Cas9-gRNA expres G by copy number assay. (~14d after sorting), the genomic DNA from coloniesthose was characterized 96-well plates by manual picking. Once individual iPSC colonies were available (2–3 d after sorting), they were collected into individual wells of Matrigel-coated nozzle under sterile conditions. Once multicellular colonies were clearly visible well of Matrigel-coated 96-well plates by a BD FACSAriaII sorter with a 100- GFP before being sorted. After adding the viability dye TO-PRO-3 (Invitrogen), the rene tubes with 35- inhibitor (Santa Cruz Biotech). All samples were filtered through 5-mL polysty single-cell suspension with Accutase and resuspended in PBS with 10 isolated were by FACS. cells At 72 h after nucleofection, single the iPSCs were dissociated treatment, into a CRISPR/Cas9 following colonies iPSC isogenic Single-cell isolation by fluorescence-activated cell sorting (FA following sequences: the with IDT by synthesized were primers The PCR. nested by amplified was r 16p11.2 in Forisolation sequences,the genomicthe region around CRISPRthe target sequences site junction the of Identification and a newly designed reverse primer targeting the unique rMDS segment. the PCR was performed using the same forward primer as in detectiontion to amplify of part ofdeletion the 16p11.2 region that is deleted in 16p11.2 rMDS. Also, iPSC lines, we followed the same PCR protocol performedFig. 2b in the detection of dele the exact modification that occurred (for the 575-kb deletions) ( products were visualized, which was followed by Sanger sequencing to for°C95 for °Cdetermine63s,15 for °C 72 s, 30min (45 1cycles); for°C 72min. 2PCR Fidelity Master Mix (NEB), with the following cycling conditions: 95 °C for 2 min; Technologies (IDT) with the following sequences: ( tion, the genomic region flanking the CRISPR target site was amplified by PCR preparedRapidDNAusingtheby method extraction colonies, iPSCs were detached with ReLeSR (Stem Cell Technologies) and then Screening of individual iPS six sets of TaqManof microdeletion sets region16p11.2atsix genes probestargeting six number screening of CRISPR-treated iPSC lines, qRT-PCR was performed using C tion that occurred ( (ThermoFisher Scientific) and Sanger sequencing to determine the exact muta productsPCR weremin.for visualized,followed 1 Blunt byZerocloning PCR 95 °C for 2 min; 95 °C for 15 s, 61 °C for 30 s, 72 °C for 1 min (45 cycles); 72 °C Phusion High Fidelity Master Mix (NEB), with the following cycling conditions:was reaction PCR performed second The using min. 1 1 for °C 72 cycles); (45 min 1 for °C 72 s, 30 for °C 61 s, 15 for °C 95 min; 2 for °C 95 conditions: cycling following the Phusionreverse-1primerswithandandFidelity Master(NEB),High Mix Supplementary Fig. 2a Fig. Supplementary opy number analysis: quantitative real-time P enomic PCR reactions were performed using 2 The first PCR reactionThefirstPCR performed was using 1 + Reverse, 5 Forward, 5 Reverse-2, 5 Reverse-1, 5 Forward, 5 TO-PRO-3 ). To determine the heterozygosity of 16p11.2 region in CRISPR targeting DN A extraction.A ′ -CCCTCAGGTCATCCTCTCAT-3 ′ ′ -CAACTGCAATTTCCTTTTCAA-3 -TGCAAGTTTCAGGAAACTTGG-3 – ′ ′ iPSCs were sorted and plated, with one cell placed in each in placed cell one with plated, and sorted were iPSCs -GTAGAGGCGGGCTGGCCAG-3 -CGTGGTGGGAGACATGCACCA-3 µ µ Supplementary Fig. 5 l of the first PCR product, forward and reverse-2 primers and m mesh cell strainer caps (BD Falcon 352235) immediately ). The primers were synthesized by Integrated DNAIntegrated by synthesized were primers The ). For high-throughput 96-wellextractioninformat, C colonies. To isolate genomic DNA from the iPSC µ ). l of genomic DNA and Phusion High C R (qR µ nature nature l ofgenomicl DNA, forward ′ ′ 3 ′ 5 T ′ . Fordeleofdetection. -P ′ C 3 MD NEUR 5 R). . Cells were Cells lysed . Supplementary For initial copy C iPS S S). OSCI µ To obtain M ROCK C lines. EN µ C m E - - - - -

© 2016 Nature America, Inc. All rights reserved. ( family) (one microduplication 16p11.2 with patients from derived lines 3 as well as families) (two microdeletion 16p11.2 harboringpatients from derived prepared( linesiPSC from23 L ments (including chromosomal translocations, insertions and inversions).uniparental disomy, methylation abnormalities or other chromosomaleffective rearrange resolution. The assay is also not specificallyhg19. designedThis assayto detectdoes notmosaicism, exclude chromosome anomalies smallerhgGateway than the assay’s of the Human Genome (UCSC Genome Browser, oligonucleotide information is based on the March 2006, NCBI 36.3 (hg18) buildminimum average log ratio of 0.25 for genome.one-copy A genomic gains imbalance andis noted–0.50 when forsix orone-copy more oligonucleotidesThis array platformlosses; contains 180,880show probes taken a from throughout thehuman control DNA sample obtained from a pool of karyotypically genomicnormal DNAindividual(s). sample being tested by comparing the test manufacturer.the DNA toassayThe tests forimbalances a (thatPromegagainsis, losses)orthe in male CGH Microarray (design number 022060) according to the protocol providedSurePrint180KHuman × G3 Agilent byizationperformedthe4 wason (aCGH) C for multiple comparisons. using one-way analysis of variance (ANOVA) followed by Dunnett’s adjustment experiments are in triplicate. Statistical analysis for genecopy reference numberand gene was test performed the for values threshold was 2 determinedbyusingthe genes of number copy Relative s. 10 for °C 40 step, cooling cycles); (50 s 1 for °C 72 and s 30 for °C 55 s, 10 for °C amplification95step, min; 10 for system (Roche) under the following cycling conditions: preincubation step, 95 °C Hs03893363_cn. Hs00989444_cn; with the following sequences: The 16p11.2-region primers and FAM-labeled probes were synthesized by IDT DNA sample, LightCycler 480 Probes Master (Roche) and 2020× triplicatein inout carriedwere primer/probe mix. (TaqMan Copy Number Reference Assay RNase P,and a reference primer/probe Appliedset targeting the Biosystems). Reactions nature nature with or without guide RNAs. All RNA samples were extracted with Trizol reagent Scientific, with the following assay identifiers: targeting set primer/probe the reference a and SDs targeting CRISPR 15q13.3 ( genes two TaqMantargeting ofprobes sets two TRPM1 performed using six sets of TaqMan probes targeting six genes ( Hs03916555_cn; Supplementary Fig. 3 SupplementaryFig. bay rprto ad R and preparation ibrary hromosomal microarray analysis.

Amplification was performed by using the LightCycler 480 Real Time PCR Time Real LightCycler480 the using by Amplification performed was Forthe copy number screening in the 15q13.3 CRISPR work, qRT-PCR was RPPH1 KCTD13-probe, 5 KCTD13-R, 5 KCTD13-F, 5 QPRT-probe, 5 QPRT-R, 5 QPRT-F, 5 SULT1A4-probe, 5 SULT1A4-R, 5 SULT1A4-F, 5 SLX1A-probe, 5 SLX1A-R, 5 SLX1A-F, 5 BOLA2-probe, 5 BOLA2-R, 5 BOLA2-F, 5 CDIPT-probe, 5 CDIPT-R, 5 CDIPT-F, 5 , KLF13 NEUR / gene. All primer/probe sets were purchased from ThermoFisher from purchased were sets primer/probe All gene. ).However, thedata reports were generated using thegenome build ′ , ′ -CTAAACCGGAAGAGGATGACAC-3 OSCI ′ ′ OTUD7A -CATTGGCCACTGACCCTAAA-3 ′ -AGGCTGCCTCCGGAT AG-3 ′ ′ CHRNA7 TRPM1 -GGCCCTGAGCCAAGAATATC-3 -GGTTGATTGCCGTGACCTA-3 ′ -ATCAGAGCCTGAGGAAGGT-3 -AAGCCATCCACAGCCTTC-3 -TGGAGGCGGGTGTAGAA-3 ′ ′ -CTCAGACTGTGGTGATGTCAG-3 ′ -CCAGCTGGTTAAGAGGGATTTA-3 ′ -AGCCTGTATTGGAAAGGAAGAG-3 ′ -TTACTTCTCCAAACCCTTCTCC-3 -CATGGGTTACGTGGCTCCTCAGG-3 ′ ′ -CCCGAGAGCTTGTTCCGAAGCA-3 ′ -CCAAGTCAAATAGTGAAGTCCCGCCA-3 EN -TACCATTCAGCATCGGCTCCGC-3 ′ ). Other lines were created using CRISPR/Cas9 systemCRISPR/Cas9 createdusing were lines Other ). -CACACTTTGGCATGGACGATGCAC-3 ′ -TCTGAGCTGAAAGAGTGAATGCCCG-3 , Hs00942380_cn; C and – , Hs03904338_cn; E ∆ ∆ N Ct Supplementary3 Fig. -eunig (R A-sequencing CHRNA7 method µ Array-based comparative genomic hybrid l total volumecontainingtotal l 1 3 ) in the 15q13.3 microdeletion region, 6 , where, KLF13 FAN1 SCG5 RPPH1 h ttp://genome.ucsc.ed N ′ ∆ SCG5 , Hs02255264_cn; ′ , Hs07226355_cn; , Hs03900067_cn; and ′ Aseq). Ct is the differencethecycle of Ctis ′ ). There were 5 iPSC linesiPSC There were).5 ′ ′ gene on chromosome 14 ′ RPPH1 and ′ ′ N smls were samples RNA ′ ′ ′ TJP1 FAN1 ′ ′ . For each gene, each For . ′ µ ) outside the the outside ) l of genomicof l ′ , MTMR10 MTMR10 OTUD7A ′ u/cgi-bin/ ′ TJP1 - - , , , ,

assessed based on contrasts for hypothesis testing using both nominal and FDR models (GLMs) assuming a negative binomial distribution, and significance was scripts. Read counts were analyzed in the tran spike-inR ERCC environmentthe for length minimum the is usingwhich length, generalizedin nt <250 linear Wesize.library bythe filteredoutrRNA also andtRNAgenesaswell genesas >3 reads across all samples were chosen for subsequent analysis and normalized old genes within the lower limits of detection (described in ref. strand specificity (described in ref. SuiteBedTools the using quantified were counts Read namely, PicardTools, the RNAseqQCutilities;multiple for wrappers script custom with performed was alignments human reference using GSNAP v 2014/12/19 (ref. babraham.ac.uk/proje includes quality control of fastq reads using FastQc ( R 50-cycle reads for each sample. lanes of an Illumina HiSeq2500, generating an average of 40 million paired-end stream analyses toallow validation ofresponse dose and fidelitythe of procedurethe downin cover a 10 RNAssynthetic These concentrationsequence. knownand standardsRNAof 1:100 dilution of ERCC RNA Control Spike-Ins (Ambion) containing 92 synthetic 42. 41. 40. 39. 38. 37. 36. 35. 34. 33. 32. multiple comparisons. Full-length blots are shown in using one-way analysis of variance (ANOVA)usingNIH Image followedJSoftware. Statistical analysis byfor western blot Dunnett’swas performed adjustment for Healthcare) for visualization of protein bands. The protein bands cencewere HRP quantifiedsubstrate (Millipore) and exposed to Amersham Hyperfilm ECL (GE ab6721, Abcam). The membrane was developed by reacting with chemilumines no. ab97250, Abcam) and HRP-conjugated goat-anti rabbit IgGondary antibodyantibodies (cat. used no.were HRP-conjugated goat-anti mouse IgGThermoFisher antibody Scientific),(cat. rabbit anti- using the following antibodies:by conductedmouse was anti-MAPK3analysis blot western(12D11, and Sartorius)cat.11306-41BL, no.no. (cat.MA1-13041, proteinsresolved bySDS-PAGE weretransferred nitrocelluloseato membrane Bis–TrisNuPAGE10%The Scientific). (ThermoFisherby resolved was lysate 20 mg/ml leupeptin, 20 mg/ml aprotinin, 50 mM NaF and 1 mM Na IGEPALCA-630,phenylmethylsulfonylmM 1 fluoride,mg/mlpepstatin 20 A, EDTA,1% mM 2 NaCl, mM 150 7.4),Tris-HCl (pH mM (50 RIPAbuffer in iPS analysis procedures, readers are referred to our previous study P Prep Kit (Illumina cat. no. RS-122-2102) Each library also included 1 included also library Each RS-122-2102) no. cat. (Illumina Kit Prep Sample mRNA Stranded TruSeq a using RNA total of ng 200 from prepared according to the manufacturer’s instruction (Invitrogen). RNAseq libraries were 31. -value cut-offs. For details of the model fitting procedures and the statistical the proceduresandfitting model the of detailsFor cut-offs. -value N A

Aseq data analysis. C una, .. Hl, .. ETos a lxbe ut o uiiis forcomparing U. Verbovšek, of utilities suite flexible a BEDTools: I.M. Hall, & experiments. A.R. RNA-seq Quinlan, of control quality RSeQC: W. Li, & S. Wang, L., Wang, and D.S. DeLuca, variants complex of detection SNP-tolerant and Fast S. Nacu, & T.D. Wu, Jiang, L. M.B. Yaffe, mouse of PCR-sexing and extraction DNA Rapid A.H. Sinclair, & P.J. McClive, P. Mali, exhaustive enumeration of and fast “Off-Spotter”: very Rigoutsos, I. & Pliatsika, V. counting parallel efficient for approach lock-free fast, A C. Kingsford, & G. Marçais, Ran, F.A.Ran, to be overexpressed in glioblastoma. in overexpressed be to features. genomic Bioinformatics optimization. reads. short in splicing 21 pathways. signaling of prediction embryos. (2013). 823–826 genomiclookalikes fordesigning CRISPR/Cas guideRNAs. k-mers. of occurrences of 8 , 2281–2308 (2013). 2281–2308 , Supplementary whole-cell lysate preparation and western blot. , 1543–1551 (2011). 1543–1551 , 6 range of concentration, as well as varying in length and GC content t al. et et al. Mol. Reprod. Dev. Reprod. Mol. et al. et 3 t al. et Bioinformatics 7 Synthetic spike-in standards for RNA-seq experiments.

t al. et N-udd ua gnm egneig i Cas9. via engineering genome human RNA-guided et al. et . Libraries were multiplexed, pooled and sequenced on multiple 28 Genome engineering using the CRISPR-Cas9 system. CRISPR-Cas9 the using engineering Genome , 2184–2185 (2012). 2184–2185 , Bioinformatics A motif-based profile scanning approach for genome-wide approach scanning profile motif-based A M RNAseq data was processed using a standard workflow, which N-eC RAsq erc fr ult cnrl n process and control quality for metrics RNA-seq RNA-SeQC: Expression analysis of all protease genes reveals cathepsin K cathepsin reveals genes protease all of analysis Expression cts/fastqc ethods Bioinformatics Bioinformatics

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