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WO 2017/184227 A2 26 October 2017 (26.10.2017) W !P O PCT

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WO 2017/184227 A2 26 October 2017 (26.10.2017) W !P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/184227 A2 26 October 2017 (26.10.2017) W !P O PCT (51) International Patent Classification: bridge, Massachusetts 02139 (US). RIOS, Xavier; 368 C12N 15/70 (2006.01) Riverway, Apt. 7, Boston, Massachusetts 021 15 (US). (21) International Application Number: (74) Agent: IWANICKI, John P.; Banner & Witcoff, Ltd., 28 PCT/US201 7/016 184 State Street, Suite 1800, Boston, Massachusetts 02109 (US). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 02 February 2017 (02.02.2017) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (25) Filing Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (26) Publication Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, (30) Priority Data: KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, 62/291,499 04 February 2016 (04.02.2016) US MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 62/3 15,336 30 March 2016 (30.03.2016) US PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (71) Applicant: PRESIDENT AND FELLOWS OF HAR¬ SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, TR, VARD COLLEGE [US/US]; 17 Quincy Street, Cam TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. bridge, Massachusetts 02138 (US). (84) Designated States (unless otherwise indicated, for every (72) Inventors: CHURCH, George M.; 218 Kent Street, kind of regional protection available): ARIPO (BW, GH, Brookline, Massachusetts 02446 (US). GREGG, Christo¬ GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, pher J.; 19 Walter Street, Roslindale, Massachusetts 02 131 UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (US). LAJOIE, Marc J.; 308 Brookline Street, #1B, Cam TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (54) Title: RECOMBINASE GENOME EDITING Γ ΤΤ 1 r IV < Enriched SEER Library Deep Sequencing 00 (57) Abstract: A method of altering a target nucleic acid sequence within a cell is provided including providing the cell with a donor nucleic acid, providing the cell with a single strand annealing protein, and providing the cell with a single strand DNA binding protein, wherein one or more or both of the single strand annealing protein and the single strand DNA binding protein is foreign to the cell, and o wherein the donor nucleic acid is recombined into the target nucleic acid. o [Continued on nextpage] WO 2017/184227 A2 llll II II 11III I II I II I III II I II III II I II EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: — without international search report and to be republished upon receipt of that report (Rule 48.2(g)) — with sequence listing part of description (Rule 5.2(a)) RECOMBINASE GENOME EDITING RELATED APPLICATION DATA This application claims priority to U.S. Provisional Application No. 62/291,499 filed on February 4, 2016 and to U.S. Provisional Application No. 62/315,336 filed on March 30, 2016 which are hereby incorporated herein by reference in their entirety for all purposes. STATEMENT OF GOVERNMENT INTERESTS This invention was made with government support under DE-FG02-02ER63445 awarded by the Department of Energy. The government has certain rights in the invention. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 2, 2017, is named 010498_00906-WO_SL.txt and is 161,995 bytes in size. FIELD The present invention relates in general to genome editing methods that use foreign recombinases. BACKGROUND Oligonucleotide-mediated recombination is used for genome engineering (see Carr and Church 2009) where mutation-encoding oligonucleotides modify the genome without the need for site-specific DNA-binding proteins. Such techniques may be used to generate large complex libraries of genetic variants. Such techniques may also be used to generate complex, user-defined genotypes at high efficiency in E. coli. (see Wang et al., 2009). Oligo-mediated recombination has enabled multiple synthetic biology applications such as genetically recoded organisms (Lajoie et al., 2013) and sensor-based metabolic pathway optimization (Raman et al., 2014). Efforts at recombineering (i.e., recombination-mediated genetic engineering) ( are present in (VanPijkeren and Britton, 2012), (van Pijkeren et al., 2012), (van Kessel and Hatfull, 2007), (Binder et al., 2013), (Datta et al., 2008). In E. coli, oligo-mediated targeting is most commonly done via λ Red recombineering, where an oligo preferentially anneals to the lagging strand of the genome during DNA replication and incorporates into the daughter strand (Ellis et al., 2001a). This system is based on the phage λ Red operon normally expressed during the phage's lytic growth (Poteete, 2001) and promotes high-efficiency, targeted recombination between linear, single-stranded (Mosberg et al., 2010) DNA (ssDNA) and the host chromosome. The λ Red operon is composed of Red α, β and γ , also known as exo (a 5' - 3' exonuclease), beta (a single stranded annealing protein [SSAP]), and gam (a RecBCD nuclease complex inhibitor), respectively λ β is necessary and sufficient to recombine ssDNA into the E. coli chromosome and itself improves recombination rates in by lE4-fold (Ellis et al., 2001b). The β-mediated recombination is based on the input ssDNAboth directing proper targeting and encoding mutations of interest. SUMMARY The disclosure provides methods of optimizing genome editing in organisms, such as bacteria. The disclosure provides for the identification of recombinases that can be used for genome editing in organisms, such as bacteria. A recombinase may also be referred to herein as a single strand annealing protein. Genome editing includes the use of a recombinase to recombine genomic DNA to include a donor nucleic acid sequence such as a single stranded DNA (ssDNA). Such genome editing may be known in the art as "recombineering." The disclosure provides for the identification and use of components sufficient to produce introduction of a foreign nucleic acid sequence into the genome of a cell. One or more or all of such components may be foreign to the cell. Such components include a recombinase (also referred to as a single strand annealing protein or SSAP) and a single-strand binding protein. The disclosure provides for the identification of one or more pairs of a recombinase and a single-stranded binding protein that can be used in genome editing to incorporate an ssDNA into a genome. A single stranded binding protein (SSB) or a single stranded annealing protein (SSAP) is one that participates in replication, repair or recombination. An exemplary recombinase used for recombineering is λ Red as described in (Carr et al., 2012; Lajoie et al., 2012; Miki et al., 2008; Mosberg et al., 2012; Wang et al., 2009, 2011). An exemplary single- stranded binding protein is single-strand DNA-binding protein (SSB), an example of which is found in E. coli. See Meyer RR, Laine PS (December 1990), Microbiol. Rev. 54 (4): 342-80. Other exemplary recombinases or single-strand DNA-binding proteins may be found in other bacteria and viruses. The disclosure provides that either one or both of a recombinase and a corresponding single-stranded binding protein is foreign to the organism which uses them for genome editing or into which they are provided. According to one aspect, the recombinase and a corresponding single-stranded binding protein are provided to a cell as native species or as a nucleic acid encoding the recombinase or the corresponding single-stranded binding protein for expression within the cell. The disclosure provides a method of genome editing by including one or more or both of a recombinase and a corresponding single-stranded DNA- binding protein into a cell where one or more or both of a recombinase and a corresponding single-stranded DNA binding protein is foreign to the cell and where a donor nucleic acid sequence is introduced into the genome of the cell. The disclosure provides that the combination of a recombinase and a corresponding single-stranded DNA binding protein provide the minimal functional units used by a cell to insert ssDNA into its genome. The recombinase and a corresponding single-stranded DNA binding protein may be evolved from the same or different organisms. However, at least one is foreign to the cell into which they are provided or are otherwise present. The disclosure provides a library-based method of identifying candidate single- stranded annealing proteins for use in oligo-recombination. The disclosure provides a library- based method of identifying candidate single-stranded annealing proteins from various and diverse organisms for use in oligo-recombination. The disclosure provides a method by which β anneals complementary ssDNA pre- coated with SSB which is dependent on the C-terminal 8 amino acid tail of SSB. The disclosure provides a method by which the C-terminus of λ β is involved in its interaction with SSB. The disclosure provides a method of co-expressing a low-activity SSAP and its corresponding SSB to achieve oligo recombination.
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