WO2017015101A1 - Procédés de maximisation de l'efficacité de correction de gène cible - Google Patents

Procédés de maximisation de l'efficacité de correction de gène cible Download PDF

Info

Publication number
WO2017015101A1
WO2017015101A1 PCT/US2016/042463 US2016042463W WO2017015101A1 WO 2017015101 A1 WO2017015101 A1 WO 2017015101A1 US 2016042463 W US2016042463 W US 2016042463W WO 2017015101 A1 WO2017015101 A1 WO 2017015101A1
Authority
WO
WIPO (PCT)
Prior art keywords
donor
nucleic acid
sequence
target
hdr
Prior art date
Application number
PCT/US2016/042463
Other languages
English (en)
Inventor
Nancy Maizels
Luther DAVIS
Original Assignee
University Of Washington
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Washington filed Critical University Of Washington
Publication of WO2017015101A1 publication Critical patent/WO2017015101A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases

Definitions

  • the present invention relates to methods of targeted genome engineering.
  • DNA nicks are the most common form of DNA damage, but the potential of DNA nicks to contribute to genomic instability was overlooked for considerable time, because nicks were presumed to undergo immediate religation.
  • This view was challenged when it became possible to compare outcomes of DNA nicks and double-strand breaks (DSBs) targeted to specific sites in genomic DNA by nickase derivatives of sequence-specific endonucleases.
  • DSBs double-strand breaks
  • Canonical HDR occurs at DSBs, generally uses a double stranded (ds) donor nucleic acid, and requires the protein factors BRCA2 and RAD51.
  • Alternative HDR is suppressed by BRCA2 and RAD51.
  • alternative HDR can be stimulated by inhibition of BRCA2 and/or RAD51.
  • the alternative HDR pathway efficiently uses a single-stranded (ss) DNA or nicked dsDNA as a donor.
  • the present invention relates in part to the discovery of a novel annealing-dependent strand synthesis (ADSS) pathway that supports HDR at targeted DNA nicks using single-stranded nucleic acids as a preferred donor.
  • ADSS annealing-dependent strand synthesis
  • the inventors demonstrate that the efficiency and precise outcome of this pathway are dependent upon the donor strandedness relative to the nicked target strand, homology of the donor to the target, and the location of non-homologous sequence in the donor relative to the nick.
  • aspects of the invention relate to methods for optimized selection of sites of genomic nucleic acid targets and design of single-stranded donor nucleic acids to allow increased frequency of homology-directed modification at nicks in the selected target. Described herein are methods of designing and making a single-stranded donor nucleic acid for use in genome modification at a targeted nick. Also featured are methods for initiating homology-directed modification with higher frequency and fidelity using optimized single-stranded donor nucleic acid designed as disclosed herein for efficient targeted genome engineering and gene correction.
  • a method of designing a single -stranded donor nucleic acid for homology-directed modification of a genomic nucleic acid at a sequence of interest in a reaction involving a directed nicking enzyme that generates a nicked strand and an intact strand at a directed site in or adjacent to the sequence of interest comprising: a) designing a single-stranded donor nucleic acid comprising a target-homologous sequence element and a gene modification sequence element, wherein the target-homologous sequence element of the donor hybridizes to the nicked strand bearing the 3 ' end of the nick and the gene modification sequence element of the donor is exclusively 3' of the nick when the target-homologous sequence element of the donor nucleic acid is hybridized to the nicked strand; or b) designing a single -stranded donor nucleic acid comprising a target-homologous sequence element and a gene modification sequence element, wherein the target-
  • a method of making a single-stranded donor nucleic acid for homology-directed modification of a genomic nucleic acid at a sequence of interest in a reaction involving a directed nicking enzyme that generates a nicked strand and an intact strand at a directed site in or adjacent to the sequence of interest comprising: a) synthesizing a single-stranded donor nucleic acid comprising a target-homologous sequence element and a gene modification sequence element, wherein the target-homologous sequence element of the donor hybridizes to the nicked strand bearing the 3 ' end of the nick and the gene modification sequence element of the donor is exclusively 3 ' of the nick when the target-homologous sequence element of the donor nucleic acid is hybridized to the nicked strand; or b) synthesizing a single-stranded donor nucleic acid comprising a target-homologous sequence element and a gene modification sequence element,
  • the target-homologous sequence element of the donor hybridizes to the intact strand and the donor heterology relative to sequence on the 5 ' side of the nick is less than 7 nucleotides. In other embodiments, the target-homologous sequence element of the donor hybridizes to the intact strand and the donor heterology relative to sequence on the 5 ' side of the nick is less than 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides, or one nucleotide (i.e., no heterology). As demonstrated in the Examples herein, heterology greater than 7 nucleotides does not abolish alternative HDR.
  • heterology on the 5 ' side of the nick in target-homologous sequence elements should preferably be 7 nucleotides or less.
  • a method of homology-directed modification of a genomic nucleic acid at a sequence of interest comprising: a) making a single stranded donor nucleic acid according to the method described herein; and b) contacting a genomic target nucleic acid in a cell with: i) the single-stranded donor nucleic acid; and ii) a nicking enzyme that generates a nicked strand and an intact strand at a directed site in or adjacent to the sequence of interest; wherein the single-stranded donor replaces the genomic nucleic acid sequence at the sequence of interest via homology-directed repair, thereby effecting homology-directed modification of the genomic sequence at the sequence of interest.
  • the method further comprises treating or contacting the cell with an inhibitor of one or more of RAD51, BRCA2, PALB2 and SHFM1.
  • the method further comprises treating or contacting the cell with an agonist of BRCA1.
  • the method further comprises treating or contacting the cell with an inhibitor of one or more of RAD51 , BRCA2, PALB2 and SHFM 1 , and with an agonist of BRCA1.
  • the target-homologous sequence element of the donor hybridizes to the intact strand and the donor heterology relative to sequence on the 5 ' side of the nick is less than 7 nucleotides. In other embodiments, the target-homologous sequence element of the donor hybridizes to the intact strand and the donor heterology relative to sequence on the 5 ' side of the nick is less than 6 nucleotides, less than 5 nucleotides, less than 4 nucleotides, less than 3 nucleotides, less than 2 nucleotides, or there is no heterology (i.e., complete complementarity between the donor and the intact strand on the 5' side of the nick).
  • the nicking enzyme is a nicking variant of a Cas enzyme.
  • the Cas enzyme variant is a Cas9 enzyme variant.
  • the Cas9 enzyme variant is S. pyogenes Cas9 D10A .
  • the Cas enzyme variant has a mutation at a site selected from polypeptide sites corresponding to D 10, H840, N854, and N863 of the mature Cas9 polypeptide of S. pyogenes.
  • the Cas enzyme variant has a mutation corresponding to a mutation selected from D10A, H840A, N854A and N863A of S. pyogenes Cas9.
  • the technology described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.
  • FIGs. 1A-1D show single -stranded oligonucleotide (SSO) donor strand and target site preference of HDR.
  • FIG. 1A Diagram of HDR at a nick by a SSO donor complementary to the intact (cl) or nicked (cN) target strand. Top, nicked DNA; bottom, annealed cl (left) and cN (right) donors, with sequence to be transferred shown in gray.
  • FIG. IB Diagram of HDR at a nick by a SSO donor complementary to the intact (cl) or nicked (cN) target strand. Top, nicked DNA; bottom, annealed cl (left) and cN (right) donors, with sequence to be transferred shown in gray.
  • FIG. IB Diagram of HDR at a nick by a SSO donor complementary to the intact (cl) or nicked (cN) target strand. Top, nicked DNA; bottom, annealed cl (left) and
  • FIGs. 1C Diagram of the Traffic Light (TL) reporter used in the Examples described herein, showing the central 38 bp heterologous region; the positions of cleavage by gRNAs 1, 2, 8 and 9; and the 99 nt SSO-1 and SSO-2 donors carrying a central 17 nt region (gray) that must replace the heterologous region to permit GFP+ expression.
  • FIGs. 2A-2B show unidirectional conversion of markers via the cN pathway.
  • FIG. 2A Diagram of how Hindlll and Apol polymorphisms are incorporated into products of HDR by the pathways that promote conversion at nicks.
  • FIG. 2B Top, diagram of Hindlll (H3) and Apol (Al) site polymorphisms on donor SSO-3. Bottom, restriction cleavage analysis of products of HDR at nicks using the cl and cN pathways. Fragment sizes indicated at right, percent of each fragment cleaved shown below.
  • FIGs. 3A-3D show that HDR at DSBs by SSO donors is independent of BRCA2 and occurs predominantly via the ADSS pathway.
  • FIG. 3A Diagram of the TL reporter showing the position of DSBs targeted by gRNAs 1, 2, 8 and 9. Donors SSO-2 and SSO-1 are shown below the diagram.
  • FIG. 3B HDR frequencies at DSBs at each target/donor pair tested. HDR frequencies are shown as mean and SEM (n>6).
  • FIG. 3C Restriction cleavage analysis of conversion of Hindlll (H3) and Apol (Al) site polymorphisms in products of HDR at DSBs targeted by gl or g8 and supported by donor SSO-3.
  • FIG. 3D Postulated intermediate formed during HDR by SSO-3 at a DSB, using the ADSS pathway.
  • FIG. 4 shows a contrasting role of RAD51 in HDR at DSBs and DNA nicks.
  • a DSB undergoes 5 '-3' excision, then RAD51 loads on the exposed 3' ends to promote strand invasion.
  • a nick is not excised but unwound at its 3' end (left) or both 3' and 5' ends, (right), then RAD51 loads to promote re-annealing of the target.
  • FIG. 5 shows a diagram of a TL reporter, showing the central 38 bp heterologous region; the positions of cleavage by gRNAs 1, 2, 3 and 8; and the SSO donor carrying a central 17 nt region that must replace the heterologous region to permit GFP+ expression.
  • Target sequence is SEQ ID NO: 1.
  • ss donor sequence is SEQ ID NO: 2.
  • FIG. 5, shows stimulation of alternative HDR upon inhibition of BRCA2.
  • FIG. 6 shows that limiting heterology at the 3' end of nick promotes cN donor use.
  • FIG. 7 shows that cN conversion tracts extend 3', but not 5' from the nick.
  • FIG. 8 illustrates a proposed model for alternative HDR using a cN donor and cl donor.
  • FIG. 9 provides sequence conversion diagrams for alternative HDR at a nick and the elucidated guidelines to optimize donor selection.
  • Described herein are methods to optimize targeted homology-directed genomic modification.
  • methods are described to optimize targeted homology-directed genomic modification at the site of a single -stranded nick.
  • aspects of the invention relate to methods for optimized selection of genomic nucleic acid targets and design of single-stranded donor nucleic acids to allow increased frequency of homology-directed modification at nicks in the selected target via hybridization of the donor to the nicked strand (cN pathway) or intact strand (cl pathway) at targeted nicks in the selected region.
  • methods of making a single -stranded donor nucleic acid for use in genome modification at a targeted nick Further embodiments include methods for initiating efficient homology-directed modification at a targeted nick within a genomic region of interest, using a single -stranded donor nucleic acid as disclosed herein.
  • NHEJ Non-Homologous End-Joining
  • HDR Homology-Directed Repair
  • HDR "Homology-Directed Repair” involves a donor with homology to the genomic region subject to repair - the process uses a "donor” molecule to direct repair of a "target” molecule, and can lead to the transfer of genetic information from the donor to the target.
  • HDR can occur at nicks or DSBs and can result in the alteration of the sequence of the target molecule (e.g., insertion, deletion, mutation, including site-directed mutation) if the donor nucleic acid molecule differs from the target molecule and part or all of the sequence of the donor molecule is incorporated into the target DNA.
  • Canonical HDR requires BRCA2 and RAD51, and repairs a DSB, employing a double -stranded DNA (dsDNA) donor molecule.
  • alternative HDR is an HDR mechanism that is suppressed by BRCA2, RAD51, and functionally -related genes (see, e.g. Table 5), and that repairs a DNA nick, using a ssDNA or nicked dsDNA donor molecule.
  • alternative HDR is positively regulated by BRCA1 and/or requires BRCA1. The nicks that initiate alternative HDR induce less local mutagenesis than the DSBs that initiate canonical HDR, which suits alternative HDR well for genomic engineering.
  • nicking enzymes also called “nickases” - can be targeted to nick genomic DNA at or near essentially any site (e.g., through the use of guide RNAs), though local sequence context may influence the precise site of the nick.
  • the discovery of the influence of single-stranded donor strandedness and modifying sequence location relative to a nick therefore permits the optimal design of donors for genomic modification at the site nicked by any given targeted nickase enzyme.
  • the single-stranded donor is complementary to the nicked strand, the donor is referred to as a "cN donor.”
  • the donor is referred to as a "cl donor.”
  • the conversion tract i.e., the tract of sequence including the change to be introduced by alternative HDR
  • the conversion tract will extend exclusively 3 ' of the nick. This reflects the fact that the 3' end of the nick primes DNA synthesis using the donor as template. Thus, if and when the donor is complementary to the nicked strand, the sequence to be modified should be entirely 3 ' of the nick;
  • an optimal donor will be completely homologous with the region 5 ' of the nick in the target genomic DNA, and the heterologous sequence used to modify the target will be exclusively 3 ' of the nick. See, for example, FIG. 9 for a schematic.
  • conversion can occur at either or both sides of the nick. If and when the donor is complementary to the intact strand, there is a preference for the region to be modified to be 5 ' of the nick, but the sequence to be modified can be on either side of the nick. That is, targeting the nick such that the region to be modified is 5 ' of the nick is preferable, but not absolutely essential;
  • a method of making a single -stranded donor nucleic acid for homology-directed modification of a genomic nucleic acid at a sequence of interest in a reaction involving a targeted nicking enzyme that generates a nicked strand and an intact strand at a targeted site in or adjacent to the sequence of interest comprising:
  • alternative HDR and methods to stimulate alternative HDR as opposed to canonical HDR are described in WO 2014/172458, which is incorporated herein by reference.
  • the use of a targeted nicking endonuclease enzyme, as opposed to a targeted endonuclease that cleaves both strands of a target DNA sequence, can promote modification via the alternative HDR pathway.
  • the introduction of a construct encoding a targeted nicking endonuclease and a donor nucleic acid, e.g., a single-stranded donor nucleic acid with the properties as described herein can promote target locus modification to incorporate the donor sequence via alternative HDR.
  • Alternative HDR can be further stimulated by inhibition of one or more of RAD51, BRCA2, PALB2 and SHFM1 and/or by an agonist of BRCA1 activity.
  • RNA-guided nicking enzymes including RNA-guided nicking enzymes, inhibitors of RAD51, BRCA2, PALB2 and SHFM1 and agonists of BRCA1 that permit the practice of the technology described herein.
  • the frequency or efficiency of alternative HDR can refer to the number or proportion of cells in a targeted population that effect a desired genomic modification via alternative HDR.
  • the frequency or efficiency for a given HDR process is "increased" if it is at least 1% greater than the frequency or efficiency for a reference HDR process.
  • the frequency is increased by at least 1.5%, at least 2.0%, at least 2.5%, at least 3.0%, at least 3.5%, at least 4.0%, at least 4.5%, at least 5.0%, at least 5.5%, at least 6.0%, at least 6.5%, at least 7.0%, at least 7.5%, at least 8.0%, at least 8.5%, at least 9.0%, at least 9.5%, at least 10.0%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15% or more.
  • the frequency is increased by at least 50%, at least 75%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40 fold or more.
  • the frequency of alternative homology -directed modification at targeted nicks is increased by using single-stranded donor nucleic acids designed as described herein. This frequency can be further increased upon inhibition of one or more of RAD51, BRCA2, SHFM1 (DSS 1) or PALB2 or by expression of BRC3 or a RAD51 variant with impaired ATPase activity.
  • donor nucleic acid refers to a single-stranded nucleic acid molecule which has been introduced (i.e. introduced into a cell) to serve as a donor for HDR and thereby for homology-directed gene modification.
  • a donor nucleic acid molecule can comprise a modification to be introduced into the target cell, e.g. , at a nick.
  • a donor nucleic acid molecule can comprise, e.g., DNA, RNA, or modified versions thereof, e.g. LNA.
  • Use of donor nucleic acid comprising RNA is described in the art, e.g., in Meers C et al. DNA Repair (Amst). May 16 (2016); Keskin H et al. Nature. 515(7527):436-9; Nov 20 (2014); Storici F et al. 447(7142):338-41; May 17 (2007).
  • a single-stranded donor nucleic acid for use in the methods described herein will carry a target-homologous sequence element and a gene modification sequence element.
  • the target-homologous sequence element will include sequence sufficient in length to permit hybridization of the donor to the target DNA via the homologous sequence element.
  • the homologous sequence element in the donor will be complementary to 20 or more nucleotides of the target if there is no heterology, and to longer regions of the target if the homologous sequence element is interrupted by heterologous base pairs or heterologous sequence elements.
  • the length and degree of sequence homology both contribute to the efficiency with which the donor anneals to the target, that the length and degree of sequence homology must be sufficient to permit annealing of the target homologous sequence element of the donor to the target DNA, and that annealing will depend upon the lengths of the homologous and heterologous elements, and on the base composition, and may be further affected by incorporation of bases other than the four naturally-occurring DNA bases (A, C, G or T) that have been chemically modified to improve annealing.
  • bases other than the four naturally-occurring DNA bases A, C, G or T
  • oligonucleotide donors in the range of 80-120 nucleotides with homology blocks of at least 35 nucleotides are therefore currently practical choices for many purposes.
  • longer single-stranded donor oligonucleotides they can be produced, for example, by primer extension.
  • the desired sequence can be subcloned into a phage vector, e.g., a bacteriophage M13 vector, that packages its genome into phage particles as a single strand.
  • Longer RNA donors can be produced, for example, by runoff transcription from a DNA template.
  • a donor nucleic acid may require a tract of target homologous sequence on either side of a gene modification sequence element.
  • complementarity can be similar on both sides of the gene modification sequence element, but the lengths and degree of complementarity can also differ as long as one keeps in mind the criteria or rules provided herein regarding the location of heterology and strandedness relative to the nick.
  • the gene modification sequence element of a single-stranded donor nucleic acid for use in the methods described herein will be located 3 ' of a target-homologous sequence (and can optionally be located between two target-homologous sequences), and will differ from the target sequence in the manner by which one wishes to modify the genome.
  • the gene modification sequence element can include an insertion, deletion or change of one or more bases relative to the target sequence.
  • the change to be introduced is small, e.g., introduction or correction of a point mutation
  • the gene modification sequence element can be relatively short, including as little as one nucleotide that differs from the target sequence.
  • the change can introduce new sequence encoding one or more polypeptides or portions thereof, including, for example, a protein or peptide tag, a reporter polypeptide, a negative regulator, such as an siRNA or antisense RNA expression cassette, among others, or a genetic regulatory element (e.g., that renders one or more genes susceptible to inducible control).
  • the change can also disrupt a gene sequence, e.g., by insertional mutagenesis within a coding or regulatory portion of a target gene, or the change can excise all or a portion of a gene sequence. Insertion of a reporter expression cassette can be used to interrupt a target gene while at the same time providing an easy screen for cells that have incorporated the change on the basis of the reporter expression.
  • a gene modification can introduce a detectable "tag" to an existing or endogenous gene present in the target nucleic acid. Because of the lower rates of mutagenesis caused by the methods described herein, they are particularly well suited to carry out such modifications.
  • Detectable tags are nucleic acid sequences which generate or permit the generation of a detectable signal (e.g. by catalyzing a reaction converting a compound to a detectable product) either as a transcribed nucleic acid product or as a translated polypeptide product.
  • Detectable tags can include, by way of non-limiting example, e.g., fluorescent polypeptides (e.g. GFP; mCherry; CFP; GFP; ZsGreenl; YFP; ZsYellowl; mBanana;
  • mOrange DsRed; tdTomato; DsRed2; mStrawberry; HcRedl; mRaspberry; E2-Crimson; mPlum; Dendra 2; Timer; PAm Cherry; and Cerulean fluorescent protein
  • epitope tags e.g. HA, FLAG, V5, VSV-G, HSV, biotin, Myc, or TRX.
  • the modification can delete a region of sequence.
  • the donor can include target-homologous sequence elements that are homologous to target sequence on either side of the region to be deleted, but omitting the sequence to be deleted. HDR will result in the removal of the region missing from the donor relative to the target region.
  • a gene modification sequence element will necessarily differ from the sequence at a target sequence of interest in the genome, and will be at least 1 nucleotide in length, at least 10 nucleotides in length, at least 20 nucleotides in length, at least 30 nucleotides in length, at least 40 nucleotides in length, at least 50 nucleotides in length, at least 100 nucleotides in length, at least 200 nucleotides in length, at least 300 nucleotides in length, at least 400 nucleotides in length, at least 500 nucleotides in length or more, e.g., 1000 nucleotides or more.
  • the gene modification sequence element can be between 1 and 1000 nucleotides, for example, between 1 and 500 nucleotides, between 1 and 400 nucleotides, between 1 and 300 nucleotides, between 1 and 200 nucleotides, or between 1 and 100 nucleotides.
  • a nicking enzyme can produce a nick.
  • the site at which a targeted nicking enzyme creates a nick can be determined for a given enzyme on the basis of review of the sequence in the area of the desired change relative to the sequence constraints of the targeted nicking enzyme.
  • the Cas9 enzyme employed for DNA cleavage in the CRISPR gene-editing process requires that the genomic sequence cleaved be preceded 3 to 4 nucleotides upstream by a protospacer adjacent motif (PAM).
  • the canonical PAM (SEQ ID NO: 3) is 5'-NGG-3', where N is any of G, A, T or C.
  • the Cas9 nuclease (or a nickase variant thereof; see below) can be targeted very precisely using a heterologous guide RNA (gRNA) with homology to a given target
  • gRNA heterologous guide RNA
  • the sequence 5'-NGG-3' occurs frequently in the genome, but the exact site of cleavage is still dependent upon where near a desired target the PAM occurs.
  • the local sequence constraints for other RNA-guided nucleases/nickases are known to those of skill in the art, such that the ordinarily skilled artisan can identify, for a given nuclease or nicking enzyme, where in or near a given target the enzyme of choice will cut or nick the genomic DNA.
  • the criteria or rules described herein can be applied to design a donor nucleic acid, e.g., a single -stranded donor nucleic acid, that will mediate efficient alternative HDR to thereby introduce a desired modification to the genomic DNA.
  • a donor complementary to the nicked strand can be designed in which the conversion tract (also referred to herein as the "gene modification sequence element," which includes sequence one wishes to introduce to the genome at the selected site) extends exclusively 3 ' of the nick, i.e., such that there is no heterologous sequence 5' of the nick. See, e.g., FIG. 9, top.
  • the conversion tract also referred to herein as the "gene modification sequence element”
  • such a donor will also be designed to be completely homologous with - i.e., fully complementary to - the region in the target that is 5 ' of the nick.
  • a donor complementary to the intact strand cl donor
  • conversion can occur at either or both sides of the nick.
  • the region to be modified is 5 ' of the nick, and an optimal donor will carry limited heterology with the target on the 3 ' side of the nick. See, e.g., FIG. 9, bottom.
  • the donor nucleic acid molecule can be at least about 50 nt in length. In further embodiments, the donor nucleic acid molecule can be at least about 60 nt in length, at least about 70 nt in length, at least about 100 nt in length, at least about 200 nt in length, at least about 300 nt in length, at least about 400 nt in length, at least about 500 nt in length, at least about 1 kb in length, at least about 2kb in length, at least about 3 kb in length, at least about 4 kb in length, or at least about 5 kb in length. In some embodiments, the donor nucleic acid molecule can be from about 50 nt to about 1000 nt in length. In some
  • the donor nucleic acid molecule can be from about 50 nt to about 500 nt in length. In some embodiments, the donor nucleic acid molecule can be from about 50 nt to about 200 nt in length.
  • limited heterology means that the target-homologous sequence element of a single -stranded donor molecule is at least 85% homologous to the target sequence, e.g., at least 90% homologous (10% heterologous or less), at least 91% homologous (9% heterologous or less), at least 92% homologous (8% heterologous or less), at least 93% homologous (7% heterologous or less), at least 94% homologous (6% heterologous or less), at least 95% homologous (5% heterologous or less), at least 96% homologous (4% heterologous or less), at least 97% homologous (3% heterologous or less), at least 98% homologous (2% heterologous or less), at least 99% homologous (1% heterologous or less) or even, for example, 100% homologous (no heterology).
  • the donor and target are necessarily heterologous - i.e., they include sequence differences one wishes to introduce to the target sequence, the differences are referred to in terms of homology and heterology.
  • heterology and homology are equivalent to the degree of non-complementarity and complementarity, respectively, between the target and the "target homologous sequence element" of the donor.
  • the Examples herein demonstrate that, for example, limiting donor heterology 5 ' of the nick (i.e., at the 3' end of the nicked strand) promotes cN donor use. See, e.g., FIG. 6.
  • nickases refers to a nuclease which cleaves only one strand of a dsDNA molecule, thereby generating a nick.
  • Non-limiting examples of nickases can include a nuclease with one active site disabled; I-Anil with one active site disabled; or Cas9D10A, among others.
  • any of a number of nucleases that use an RNA or other nucleic acid guide molecule to determine the site of cutting can be advantageously adapted.
  • the specific sequence targeted for cleavage can be modified by altering the guide RNA's targeting sequence to be complementary to the selected target.
  • Cas9-derived nucleases and nickases are targeted by means of guide nucleic acid molecules, e.g. guide RNAs, which can be engineered to hybridize specifically to a desired target nucleic acid molecule.
  • Guide RNAs for nucleic acid-guided endonucleases and ways to modify them to change substrate sequence specificity are well known in the art.
  • zinc finger nucleases can be targeted by a combinatorial assembly of multiple zinc finger domains with known DNA triplet specificities.
  • Such targeting approaches are known in the art and described, e.g. in Silva et al. Curr Gene Ther 201111 : l l-27; Ran et al. Cell 2013 154: 1380-9; Jinek et al. Science 2013 337:816-821; Carlson et al. PNAS 212 109: 17382-7, Guerts et al. Science 2009 325:433-3; Takasu et al. Insect Biochem Mol Biol2010 40:759-765; and Watanabe et al. Nat. Commun. 2012 3; each of which is incorporated by reference herein in its entirety.
  • variants of the RNA-guided Cas9 endonuclease are known that only nick one strand, rather than cleaving both stands.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase that only nicks at the target site.
  • D10A aspartate-to-alanine substitution
  • pyogenes is provided herein as SEQ ID NO: 4.
  • Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • H840A, N854A, and N863A For any given nickase variant of a targeted nuclease, one of skill in the art can determine which strand of the target becomes nicked. It is contemplated that mutations of other Cas enzymes at sites corresponding to the D10A mutation in Cas9, or at sites corresponding to the H840, N854 and N863 sites of Cas9 may render the normally double-strand-cleaving enzymes capable of only nicking the target substrate.
  • Corresponds to in this context is meant that in an alignment of wild-type S.
  • the given amino acid of the other Cas enzyme protein is aligned with the Cas9 wt amino acid specified herein as, e.g., D10, H840, N854, or N863.
  • Non-limiting examples of additional Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4.
  • Embodiments of the methods herein can further comprise generating a nick in the nucleic acid molecule to be modified.
  • the method can further comprise generating a nick in the transcribed strand of the nucleic acid molecule to be modified.
  • the nick in the transcribed strand is generated by contacting the nucleic acid to be modified with a nickase specific for the transcribed strand of a dsDNA.
  • transcribed strand refers to the strand of a dsDNA which serves as the template for transcription.
  • the transcribed strand may also be referred to herein by as the "template strand.”
  • template strand In a transcribable nucleic acid molecule of known sequence, one of skill in the art can readily distinguish a transcribed strand from its complement and/or by analyzing gene expression product sequences.
  • a "transcribed strand" of a nucleic acid molecule to be modified and a donor nucleic acid molecule for alternative HDR may share homology and/or complementarity but are not necessarily related and should not be conflated.
  • the nickase enzyme is generally expressed from a vector introduced to the cell targeted for genomic modification.
  • Expression vectors and methods for their introduction into cells are known to those of ordinary skill in the art and are discussed further herein below.
  • a construct encoding the nicking enzyme can be introduced into a cell, whereupon the enzyme is expressed and nicks the target sequence.
  • the nucleic acid sequence encoding the nicking enzyme is
  • codon-optimized for expression in particular cells such as eukaryotic cells.
  • the eukaryotic cells can be derived from a particular organism, such as a mammal.
  • Non-limiting examples of mammals can include human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • the nicking enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the endonuclease).
  • a nicking enzyme fusion protein can comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • protein domains that can be fused to a nicking enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • beta-glucuronidase beta-galactosidase
  • luciferase green fluorescent protein
  • GFP green fluorescent protein
  • HcRed HcRed
  • DsRed cyan fluorescent protein
  • RNA-guided endonuclease can be fused to a gene sequence encoding a protein or a fragment of a protein that binds DNA molecules or binds to other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) VP 16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • Lex A DNA binding domain (DBD) fusions Lex A DNA binding domain (DBD) fusions
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a tagged endonuclease is used to identify the location of a target sequence.
  • the method comprising contacting the nucleic acid with an inhibitor of a factor selected from Table 5.
  • the inhibitor is an inhibitor of one or more, or any combination of RAD51, BRCA2, PALB2 and SHFM1 (DSS1).
  • alternative HDR at targeted nicks is stimulated by inhibition of one or more of the expression of BRC3 and a RAD51 variant with reduced ATPase activity relative to wild-type.
  • BRC3 and a RAD51 variant with reduced ATPase activity are considered alternative HDR at targeted nicks. The following discusses these factors and ways to modify their activity.
  • BRCA2-related activity increases alternative HDR.
  • the genes/gene products of Table 5 promote BRCA2 activity. It is therefore specifically contemplated herein that inhibition of one or more (e.g., one, two, three, four, five or more) of these genes or the factors they encode can increase alternative HDR.
  • the methods described herein relate to inhibition of one or more of RAD51, BRCA2; PALB2 and/or SHFM1 or any combination thereof.
  • RAD51 refers to a protein that forms a helical nucleoprotein filament on DNA and controls the homology search and strand pairing of DNA damage repair. Sequences for RAD51 polypeptides and nucleic acids encoding them for a number of species are known in the art, e.g. human RAD51 (NCBI Gene ID: 5888) polypeptide (SEQ ID NO: 5; NCBI Ref Seq: NP 001157741) and nucleic acid (SEQ ID NO: 6; NCBI Ref Seq: NM_001164269).
  • BRCA2 refers to a tumor suppressor gene product that normally functions by binding single-stranded DNA at DNA damage sites and interacting with RAD51 to promote strand invasion. Sequences for BRCA2 polypeptides and nucleic acids encoding them for a number of species are known in the art, e.g. human BRCA2 (NCBI Gene ID: 675) polypeptide (SEQ ID NO: 7; NCBI Ref Seq: NP 000050) and nucleic acid (SEQ ID NO: 8; NCBI Ref Seq: NM_000059).
  • DSS 1 and “SHFM1” refers to a 26S proteasome complex subunit that interacts directly with BRCA2. Sequences for DSS 1 polypeptides and nucleic acids
  • PLB2 refers to a DNA-binding protein that binds to single-strand DNA and facilitates accumulation of BRCA2 at the site of DNA damage.
  • PALB2 also interacts with RAD51 to promote strand invasion. Sequences for PALB2 polypeptides and nucleic acids encoding them for a number of species are known in the art, e.g. human PALB2 (NCBI Gene ID: 79728) polypeptide (SEQ ID NO: 11; NCBI Ref Seq: NP 078951) and nucleic acid (SEQ ID NO: 12; NCBI Ref Seq: NM_024675).
  • the gene names listed in Table 5 are common names.
  • the sequences and NCBI Gene ID numbers provided for each gene listed in Table 5 are the human sequences and accessions. Homologous genes from other species may be readily identified, e.g. the identified homologs in the NCBI database, or by querying databases, e.g. via BLAST.
  • the term "inhibitor” refers to an agent which can decrease the expression and/or activity of the targeted expression product (e.g. mRNA encoding the target or a target polypeptide), e.g. by at least 10% or more, e.g. by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more.
  • the efficacy of an inhibitor of BRCA2 can be determined, e.g. by measuring the level of the expression product of BRCA2 (mRNA and/or protein) and/or the activity of BRCA2 (e.g.
  • RT-PCR can be used to determine the level of RNA
  • Western blotting with an antibody e.g. an anti-BRCA2 antibody, e.g. Cat No. ab97; Abeam; Cambridge, MA; antibodies to other factors described herein are also commercially available
  • an antibody e.g. an anti-BRCA2 antibody, e.g. Cat No. ab97; Abeam; Cambridge, MA; antibodies to other factors described herein are also commercially available
  • the HDR-influencing activity of, e.g. BRCA2, among others can be determined using methods known in the art and assays for alternative HDR described in the Examples herein.
  • the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule.
  • an inhibitor will directly bind to the targeted factor, e.g. BRCA2 or to its mRNA. In some embodiments, an inhibitor will directly result in the cleavage of the targeted factor's mRNA, e.g., via RNA interference. In some embodiments, an inhibitor can act in a competitive manner to inhibit activity of the targeted factor. In some embodiments, an inhibitor can comprise a portion of the target factor and act as a competitive or dominant negative factor for interactions normally involving the targeted factor.
  • the methods described herein can comprise treating or contacting the cell with two or more inhibitors, e.g. two inhibitors, three inhibitors, four inhibitors, or more.
  • the methods described herein can comprise treating or contacting the cell with a plurality of inhibitors, e.g. an inhibitor of RAD51 and an inhibitor of BRCA2.
  • an inhibitor can inhibit multiple targets, e.g. an antibody or other reagent with bispecificity.
  • multiple types of inhibitors can be used, e.g. an antibody reagent specific for BRCA2 and a small molecule inhibitor of RAD51.
  • an inhibitor of a gene expression product of a gene of Table 5 can be an inhibitory nucleic acid.
  • the inhibitory nucleic acid is an inhibitory RNA (iRNA). Double -stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi).
  • RNAi RNA interference
  • the inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15- 30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of the targeted mRNA transcript. The use of these iRNAs permits the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
  • iRNA refers to an agent that contains RNA, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • an iRNA as described herein effects inhibition of the expression and/or activity of a gene selected from Table 5.
  • contacting a cell with the inhibitor results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or more relative to the level without the iRNA.
  • RNA interference molecules for inhibiting expression of a given target gene, including the introduction of such molecules into cells, whether directly via, e.g., lipid complexes, or via the introduction of nucleic acid constructs encoding the RNA interference molecules (e.g., shRNAs) or their precursors, is known to those of ordinary skill in the art.
  • RNA interference molecules e.g., shRNAs
  • a great deal of information is known to those of skill in the art regarding modifications to the RNA molecule and, e.g. conjugates with various agents that promote the stability and effectiveness of RNA interference agents.
  • RNA interference agents are commercially available for a wide range of target genes.
  • an inhibitor of a gene expression product of a gene of Table 5 can be an antibody reagent specific for the respective polypeptide.
  • a BRCA2 inhibitor can be an anti-BRCA2 antibody reagent.
  • Antibodies have, historically, been viewed as unable to cross the plasma membrane. However, antibodies have been demonstrated to gain access to intracellular protein targets (see, e.g. Guo et al., Science Translational Med. 2011 3:99ra85; W02008/136774; Guo et al. Cancer Biol and Ther 2008 7:752-9; and Ferrone. Sci Transl Med 2011 3:99ps38) both in cultured cells and in vivo.
  • an "antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F( ab')2, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
  • An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody.
  • an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody reagent encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and domain antibody (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies.
  • An antibody can have the structural features of IgA, IgG, IgE, IgD, or IgM (as well as subtypes and combinations thereof).
  • Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
  • an inhibitor of a gene expression product of a gene of Table 5 can be a small molecule.
  • Small molecule inhibitors of various targets described herein are known in the art.
  • inhibitors of RAD51 can include but are not limited to IBR2; RI-1; RI-2; and B02. See, e.g., Zhu et al., EMBO Mol. Med. 5: 353-365 (2103), Budke et al., Nucleic Acids Res. 40: 7347-7317 (2012), Budke et al, J. Med. Chem. 56: 254-263 (2013), Alaqpulinsa et al., Front. Oncol. 4: 289 (2014).
  • alternative HDR is positively regulated by BRCAl .
  • BRCAl a BRCAl agonist can be used alone, or together with an inhibitor of one or more of RAD51 , BRCA2, PALB2 and/or SHFM 1 to promote alternative HDR as described herein.
  • agonist refers to any agent that increases o level and/or activity of a target gene or its gene product, e.g., BRCAl .
  • the term refers to an agent which increases the expression and/or activity of the target by at least 10% or more, e.g. by 20% or more, 30% or more, 40% or more, 50% or more, 75% or more, 100% or more, 200% or more, or 500% or more relative to the activity in the absence of the agonist.
  • Expression levels are readily measured by, e.g., RT PCR (RNA expression level) and Western blot (protein level). Activity measurement can include assays for alternative HDR as described herein and in WO
  • An agonist can include, for example, a construct or vectorthat encodes the target gene product.
  • BRCA 1 refers to a gene encoding a polypeptide with a zinc finger domain and a BRCT domain, which is involved in DNA damage repair. BRCAl binds to DNA and interacts directly with RAD51. Sequences for BRCAl polypeptides and nucleic acids for a number of species are known in the art; human BRCAl mRNA sequence is available at, e.g. SEQ ID NO: 13; NCBI Ref Seq: NM_007294.3.
  • the gene names listed in Table 5 are common names.
  • the sequences and NCBI Gene ID numbers provided for each gene listed in the table are the human sequences and accessions. Homologous genes from other species may be readily identified, e.g. the identified homologs in the NCBI database, or by querying databases, e.g. via BLAST.
  • a method of homology-directed modification of a genomic nucleic acid comprising making a single-stranded donor nucleic acid according to the methods and criteria set out herein and contacting a cell with: a) a said single-stranded donor nucleic acid, comprising the genomic modification to be made in the cell, and b) a nickase for which the donor nucleic acid was designed, that nicks in the target region.
  • the method further comprises contacting the cell with c) an inhibitor of one or more genes of Table 5 and/or with d) an agonist of BRCAl .
  • the inhibitor is an inhibitor of RAD51; BRCA2; PALB2 and/or SHFM1.
  • the cell is contacted with the inhibitor prior to contact with the single-stranded donor nucleic acid and the nickase.
  • the single-stranded donor nucleic acid is complementary to the nicked strand of the target sequence.
  • the single-stranded donor nucleic acid is complementary to the intact strand of the target sequence.
  • the single stranded donor nucleic acid molecule comprises a target homologous sequence element which hybridizes to the intact or nicked strand and a gene modification sequence element comprised of the sequence desired to be transferred to the target sequence of interest.
  • the methods provided herein involve the delivery of one or more polynucleotides, such as or one or more vectors or plasmids encoding an enzyme, factor or nucleic acid molecule, one or more transcripts thereof, one or more proteins translated therefrom, one or more inhibitory nucleic acids or expression constructs therefor, to a host cell.
  • a targeted nuclease e.g., an RNA-guided endonuclease or nickase, in combination with (and optionally complexed with) a guide sequence is delivered to a cell.
  • an inhibitor of a factor such as an inhibitor of BRCA2 or RAD51, among others, is expressed in a cell from a vector, e.g., a vector encoding an shRNA or other inhibitory RNA (iRNA), or an antibody or intrabody.
  • a factor that stimulates alternative HDR such as BRCA1
  • a factor that stimulates alternative HDR can be expressed from a construct or vector introduced to a cell.
  • Conventional viral- and non-viral-based gene transfer methods and vectors can be used to introduce nucleic acids to mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of the alternative HDR systems described herein to cells in culture, or to cells in a host organism.
  • Vectors can include, but are not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
  • Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, calcium phosphate precipitation, cationic polymer-mediated transfection, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM). Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
  • lipid: nucleic acid complexes including targeted liposomes such as immunolipid complexes
  • Boese et al. Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4, 186, 183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • the nucleic acids can be administered to a cell by means of a viral vector, including adenoviral or retroviral (e.g., lentiviral) vectors.
  • a viral vector including adenoviral or retroviral (e.g., lentiviral) vectors.
  • Exemplary methods for introducing nucleic acid compositions for use in genome modification can be found in e.g., Mali et al. "RNA-guided human genome engineering with Cas9” Science (2013) 339:823-26; Dicarlo et al. "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems" Nucleic Acids Research (2013) 7:4336-43; Esvelt etal. "Orthogonal Cas9 proteins for RNA-guided genome regulation and editing” Nat Methods (2013) 10: 1116-21; Jao et al.
  • Donor nucleic acids can be delivered by methods now standard for gene editing methods such as CRISPR gene editing.
  • the donor nucleic acid can be delivered to the nucleus of cells in culture or cells removed from an animal or a patient (ex vivo) by experimental manipulations such as peptide-facilitated uptake, electroporation, calcium chloride, micro-injection, microprojectiles or other treatments well known to those skilled in the art.
  • the donor nucleic acid can be delivered to cells or live animals simply by exposing the cells to the oligonucleotide that is included in the medium surrounding the cells, or in live animals or humans by bolus injection or continuous infusion.
  • the donor nucleic acid may also be introduced into the cell in the form of a packaging system.
  • a packaging system include DNA viruses, RNA viruses, and liposomes as used in various gene therapy approaches.
  • the single -stranded donor nucleic acids can be dissolved in a physiologically -acceptable carrier, such as an aqueous solution or are incorporated within liposomes, and the carrier or liposomes are applied to cells in culture or, alternatively, injected into the organism undergoing genetic manipulation, such as an animal undergoing gene therapy.
  • the route of injection in mammals can be intravenous. It is understood by those skilled in the art that single-stranded donor nucleic acids are taken up by cells and tissues in animals such as mice without special delivery methods, vehicles or solutions.
  • Administration of single-stranded donor nucleic acids as described herein can also be performed locally to the area in need of treatment; this is achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • Local infusion includes intradermal, subcutaneous, intranasal, and oral routes of administration.
  • Oligonucleotides can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).
  • a solution containing the donor nucleic acids can be added directly to a solution containing the DNA molecules of interest in accordance with methods known to those skilled in the art.
  • the delivery of a targeted nickase to the cell can also be accomplished by direct introduction of the nickase protein. See, for example: Ramakrishna et al., Genome Res.
  • a viral-mediated delivery mechanism can also be employed to deliver RNA interference agents to cells in vitro and in vivo as described, for example, in Xia, H. et al. (2002) Nat Biotechnol 20(10): 1006). Plasmid- or viral -mediated delivery mechanisms of shRNA can also be employed to deliver shRNAs to cells in vitro and in vivo as described in Rubinson, D.A., et al. ((2003) Nat. Genet. 33:401-406) and Stewart, S.A., et al. ((2003) RNA 9:493-501).
  • RNA interference agents e.g., siRNAs or shRNAs
  • RNA interference agents can be introduced along with components that perform one or more of the following activities: enhance uptake of the RNA interfering agents, e.g., siRNA, by the cell, inhibit annealing of single strands, stabilize single strands, or otherwise facilitate delivery to the target cell and increase inhibition of the target gene, e.g., RAD51 or BRCA2, among others.
  • the dose of the particular RNA interfering agent will be in an amount necessary to effect RNA interference, e.g., post translational gene silencing (PTGS), of the particular target gene, thereby leading to inhibition of target gene expression or inhibition of activity or level of the protein encoded by the target gene.
  • PTGS post translational gene silencing
  • NHEJ non-homologous end-joining
  • Indels insertion/deletion mutations
  • HDR homology directed repair
  • one aspect of the present invention relates to a method of homology-directed modification of a genomic nucleic acid sequence of interest and to increased efficiency or frequency thereof.
  • the method of homology-directed modification comprises making a single-stranded donor nucleic acid as described by the methods herein and contacting a genomic target nucleic acid in a cell with the said single-stranded donor nucleic acid and a nicking enzyme that generates a nicked strand and an intact strand at a directed site in or adjacent to the sequence of interest.
  • the homology-directed modification of a genomic nucleic acid sequence of interest is nick-initiated.
  • Frequency of alternative HDR refers to the percentage of recovered cells that have undergone a modification event.
  • frequency can be determined as the proportion of cells that exhibit a particular phenotype.
  • representative samples of the target genetic material can be sequenced to determine the percentage that have acquired the desired modification.
  • Efficiency of gene modification can be represented as percentage of samples comprising the target genetic material that is identified to acquire the desired modification transferred by gene modification sequence element.
  • the single-stranded donor nucleic acid may be designed to provide the desired amino acid sequence, while also providing for a restriction site which is not naturally present in the wild-type gene, nor in the defective gene. In this manner, transformed cells can be screened to identify the presence of the desired modification by restriction digestion of their DNA, which will generate a new pattern when the new restriction site is successfully introduced.
  • higher frequency of alternative HDR would translate to higher efficiency of gene modification events.
  • the initiating nick can be targeted so that the sequence to be modified is entirely 3' of the nick.
  • the nick can be targeted so that the sequence to be modified is entirely 3 ' of the nick and the donor is complementary to the nicked strand.
  • the nick can be targeted so that the sequence to be modified is entirely 3 ' of the nick and the donor which is complementary to the nicked strand comprises a target homologous sequence element with 100% homology to the sequence at 3' end of the nick.
  • the frequency of homology-directed modification increases with a decrease in heterology between the "target-homologous sequence element" of the single -stranded donor and sequence at 3' end of the nick.
  • the frequency of homology-directed modification is about 61% higher if there is no heterology relative to 1 nucleotide heterology.
  • the frequency of homology-directed modification increases with decrease in heterology between the "target-homologous sequence element" of the single -stranded donor and sequence at 5' end of the nick.
  • the frequency of homology-directed modification is about 4% with 7 nucleotide heterology, about 2% with 27 nucleotide heterology, and less than 1% when nucleotide heterology is 37 nucleotides or higher.
  • the methods provided herein can be used for homology-directed modification at DSBs in the genomic nucleic acid of interest.
  • the single -stranded donor nucleic acid for use in homology-directed modification at DSBs comprises of a "target homologous sequence element" which hybridizes to a nicked strand of the target sequence 5 ' of the nick and the "gene modification sequence element" is exclusively 3 ' of the nick.
  • the "target homologous sequence element" of the single stranded donor nucleic acid for use in homology-directed modification of double-stranded breaks hybridizes to a nicked strand of the target sequence has sequence heterology of less than 31 nucleotides, less than 30 nucleotides, less than 27 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides, less than 1 1 nucleotides, less than 10 nucleotides, less than 7 nucleotides, less than 5 nucleotides, less than 2 nucleotides, or less than 1 nucleotide relative to the target sequence 5 ' of the nick.
  • the "target homologous sequence element" of the single stranded donor nucleic acid for use in homology-directed modification of double-stranded breaks hybridizes to a nicked strand of the target sequence, and is 100% homologous to the target sequence on the 5 ' side of the nick.
  • the efficiency of gene modification can be increased if the portion of the donor nucleic acid molecule which anneals to the target nucleic acid molecule is centered at the location of the nick generated in the target nucleic acid molecule. In some embodiments, the portion of the donor nucleic acid molecule that is complementary to a strand of the target nucleic acid molecule is substantially centered with respect to the location of the nick.
  • a molecule can be substantially centered if no more than 70% of the molecule is located to either side of the reference point (e.g. the location of the nick), e.g. 70% or less, 65% or less, 60% or less, 55% or less, or about 50% of the molecule is located to either side of the reference point.
  • a portion of a molecule can be substantially centered if no more than 70% of the portion of the molecule is located to either side of the reference point (e.g. the location of the nick), e.g. 70% or less, 65% or less, 60% or less, 55% or less, or about 50% of the portion of the molecule is located to either side of the reference point.
  • the methods provided herein can be used for example for effecting gene transfer, mutation repair, and targeted mutagenesis at a specific sequence site on a native nucleic acid segment, either in cells or in a living organism.
  • the modification can be introduced as a gene therapy, e.g., to repair a mutation or defect in the DNA of a cell and/or subject. Such repairs can restore wild type and/or normal function of a gene and/or reduce harmful effects of a gene.
  • the methods of gene modification can be performed in vivo.
  • the methods of gene modification can further comprise the step of implanting the modified cell in a subject.
  • the cell can be autologous to the subject.
  • the cell can be a stem cell, e.g. a somatic stem cell, a fetal stem cell, and/or an iPS cell.
  • the modification can correct a mutation.
  • a harmful or deleterious mutation is corrected, e.g. to the wildtype sequence and/or to a benign sequence.
  • modification can introduce a mutation.
  • a mutation can provide improved function.
  • a modification introduced according to the methods described herein can cause improved cell function.
  • improved cell function refers to an increase in at least one desirable activity that increases the productivity and/or survival of the cell or contributes positively to the health of an organism comprising the cell.
  • improved cell function can include a beneficial function the cell did not previously demonstrate, or the loss of a deleterious function the cell did previously demonstrate.
  • improved function can be accomplished by, e.g., modifying a viral gene or a gene comprising a dominant negative mutation.
  • a latent viral gene e.g. HIV
  • can be modified e.g. knocked-out or disabled.
  • deletion of genomic sequences can, for example, confer resistance to viral infection.
  • CCR5 Delta-32; Estrada-Aguirre et al., Curr. HIV Res. 11 : 506-510 (2013) can confer resistance to HIV infection.
  • Another non-limiting example relates to collagen A mutations, which are often dominant negative. By specifically targeting a modification to the defective allele that prevented synthesis of proteins, collagen would become functional in the cell (e.g. a corrective modification and/or a modification which knocks out or knocks down the dominant negative allele).
  • the rate of mutagenic end-joining is not increased as a result of the method. In some embodiments of the preceding aspects, the rate of mutagenic end-joining is not altered as a result of the method, e.g. it is neither increased nor decreased by a statistically significant amount.
  • mutagenic end joining refers to any repair pathway that directly ligates the ends of nicks or DSBs and results in at least one mutation arising relative to the original sequence. Mutagenic end-joining can include, e.g., non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ).
  • the single -stranded donor nucleic acid and methods herein can be advantageously used, for example, to introduce or correct multiple point mutations. Each mutation leads to the addition, deletion or substitution of at least one base pair.
  • Such agents may, for example, be used to develop plants or animals with improved traits by rationally changing the sequence of selected genes in cultured cells.
  • Modified cells can then optionally be cloned into whole plants or animals having the altered gene. See, e.g., U.S. Pat. No. 6,046,380 and U.S. Pat. No. 5,905,185, incorporated herein by reference.
  • Targeted base pair substitution or frameshift mutations introduced by an oligonucleotide in the presence of a cell-free extract also provides a way to modify the sequence of extrachromosomal elements, including, for example, plasmids, cosmids and artificial chromosomes.
  • the donor nucleic acids described herein also simplify the production of transgenic animals having particular modified or inactivated genes. Altered animal or plant model systems such as those produced using the methods and donor nucleic acids are invaluable in determining the function of a gene and in evaluating drugs.
  • the donor nucleic acids and methods described herein can also be used for gene therapy to correct mutations causative of human diseases. Sequences of interest will frequently be associated with mutations causing diseases. These sequences may be involved with the globin genes, in sickle-cell anemia, and ⁇ -thalassemia, with the adenosine deaminase gene in severe combined immunodeficiency, etc.
  • the situations where genetic modification will be desirable include sickle cell anemia and thalassemias, as well as other genetic diseases.
  • the target gene contains a mutation that is the cause of a genetic disorder, then the donor nucleic acid and methods herein are useful for correction of the mutation that will restore the DNA sequence of the target gene to normal.
  • the target gene is an oncogene causing unregulated proliferation, such as in a cancer cell, then the donor nucleic acid and methods described herein can be used for causing a mutation that inactivates the gene and terminates or reduces the uncontrolled proliferation of the cell.
  • the donor nucleic acid and methods described herein also provide an anti-cancer approach for activating a repressor gene that has lost its ability to repress cell proliferation.
  • the donor nucleic acid and methods described herein can provide an antiviral agent when the donor nucleic acid is specific for a portion of a viral genome necessary for proper proliferation or function of the virus.
  • the donor nucleic acid and methods described herein can also be used to generate a specific mutation in a cell line or in an animal which will provide a model to study the function of the gene product. This model can also be used to test the efficacy of a potential therapeutic agent.
  • Stem cells are used in a body to replace cells that are lost by natural cell death, injury or disease.
  • the present invention can also be used for the correction and/or alteration of a gene in the pluripotent hematopoietic stem cells of humans in order to reconstitute all or part of the hematopoietic stem cell population of that individual.
  • Stem cells of a particular tissue are capable of differentiating into a variety of different pancreatic cell types (including, but not limited to, pancreatic duct cells) when induced to proliferate.
  • the method of the present invention can be used to alter a target nucleic acid (e.g., gene) in a stem cell for the repopulation of a particular tissue(s).
  • the methods described herein can be used alone or in combination with other agents or therapeutic approaches.
  • kits comprising a donor nucleic acid as described herein (e.g., made as described herein), and a nickase or a construct or vector encoding a nickase.
  • a kit can further comprise an inhibitor of a gene expression product of a gene of Table 5, and/or an agonist of BRCA1.
  • the inhibitor can be an inhibitor of RAD51 ; BRCA2; PALB2 or SHFM1.
  • the nickase can be selected from the group consisting of: a nuclease with one active site disabled; I-Anil with one active site disabled; or Cas9 D10A .
  • the inhibitor can be an inhibitory nucleic acid. In some embodiments, the inhibitor can be an antibody reagent. In some embodiments, the inhibitor can be a small molecule, including but not limited to a small molecule inhibitor of RAD51 selected from the group consisting of: IBR2; RI-1; RI-2; and B02.
  • kits described herein can optionally comprise additional components useful for performing the methods and assays described herein.
  • reagents can include, e.g. a donor nucleic acid, transfection or viral packaging reagents, cell culture media, buffer solutions, labels, and the like.
  • reagents are known to the person skilled in the art and may vary depending on the particular cells and methods or assays to be carried out.
  • the kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • an "increase" is a statistically significant increase in such level.
  • the term “complementary” refers to the hierarchy of hydrogen-bonded base pair formation preferences between the nucleotide bases G, A, T, C and U, such that when two given polynucleotides or polynucleotide sequences anneal to each other, A pairs with T and G pairs with C in DNA, and G pairs with C and A pairs with U in RNA.
  • substantially complementary refers to a nucleic acid molecule or portion thereof having at least 90% complementarity over the entire length of the molecule or portion thereof with a second nucleotide sequence, e.g. 90% complementary, 95% complementary, 98% complementary, 99% complementary, or 100% complementary.
  • substantially identical refers to a nucleic acid molecule or portion thereof having at least 90% identity over the entire length of a the molecule or portion thereof with a second nucleotide sequence, e.g. 90% identity, 95% identity, 98% identity, 99% identity, or 100% identity.
  • telomere sequence specific for a target nucleic acid refers to a level of complementarity between the donor nucleic acid molecule and the target such that there exists an annealing temperature at which the donor nucleic acid molecule will anneal to and mediate repair of the target nucleic acid and will not anneal to or mediate repair of non-target sequences present in a sample.
  • a "portion" of a nucleic acid molecule refers to contiguous set of nucleotides comprised by that molecule.
  • a portion can comprise any subset less than all nucleotides comprised by the reference nucleic acid molecule.
  • a portion can be
  • agent refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject and which mediates or causes a desired effect within the context of a method as described herein.
  • An agent can be selected from a group including but not limited to: polynucleotides; polypeptides; small molecules; and antibodies or antigen-binding fragments thereof.
  • a polynucleotide can be RNA or DNA, and can be single or double stranded, and can be selected from a group including, for example, nucleic acids and nucleic acid analogues that encode a polypeptide.
  • a polypeptide can be, but is not limited to, a naturally -occurring polypeptide, a mutated polypeptide or a fragment thereof that retains the function of interest.
  • agents include, but are not limited to a nucleic acid aptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), small organic or inorganic molecules; saccharide; oligosaccharides; polysaccharides;
  • An agent can be applied to the media, where it contacts the cell and induces its effects.
  • an agent can be intracellular as a result of introduction of a nucleic acid sequence encoding the agent into the cell and its transcription resulting in the production of the nucleic acid and/or protein within the cell.
  • the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities that mediate or cause a desired effect within the context of a method as described herein.
  • the agent is a small molecule having a chemical moiety selected, for example, from unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof.
  • Agents can be known to have a desired activity and/or property, or can be selected, on the basis of activity, from a library of diverse compounds.
  • the term “small molecule” can refer to compounds that are "natural product-like,” however, the term “small molecule” is not limited to "natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight more than about 50, but less than about 5000 Daltons (5 kD). Preferably the small molecule has a molecular weight of less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than 700 Daltons.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • Complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non- traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%) complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • Substantially complementary refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions .
  • the term "synthesizing,” when used in the context of a single -stranded donor nucleic acid, encompasses chemical synthesis of an oligonucleotide, as well as, for example, template-directed synthesis by, e.g., primer extension, or by the preparation of a single-stranded donor nucleic acid as an insert in a single-stranded bacteriophage, such as M13.
  • genomic instability refers to the loss and/or alteration of genetic material.
  • genomic instability can be a loss of heterozygosity.
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
  • a method of making a single -stranded donor nucleic acid for homology-directed modification of a genomic nucleic acid at a sequence of interest in a reaction involving a directed nicking enzyme that generates a nicked strand and an intact strand at a directed site in or adjacent to the sequence of interest comprising: a) synthesizing a single-stranded donor nucleic acid comprising a target-homologous sequence element and a gene modification sequence element, wherein the target-homologous sequence element of the donor hybridizes to the nicked strand bearing the 3' end of the nick and the gene modification sequence element of the donor is exclusively 3 ' of the nick when the target-homologous sequence element of the donor nucleic acid is hybridized to the nicked strand; or
  • a method of homology-directed modification of a genomic nucleic acid at a sequence of interest comprising:
  • nicking enzyme that generates a nicked strand and an intact strand at a directed site in or adjacent to the sequence of interest
  • the single-stranded donor replaces the genomic nucleic acid sequence at the sequence of interest via homology-directed repair, thereby effecting homology-directed modification of the genomic sequence at the sequence of interest.
  • a nick can be converted to a DSB in S phase, but the mechanism of HDR at nicks was readily distinguished from that of HDR at breaks by characteristic strand asymmetries in donor preference and in the response to transcription, which stimulates HDR at a nick on the transcribed strand but not the non-transcribed strand [2] .
  • RAD51 is essential for canonical homology dependent repair (HDR) at DSBs in mammalian cells.
  • HDR homology dependent repair
  • 5 '-3' excision exposes single-stranded 3' ends
  • RPA coats the single-stranded DNA
  • BRCA2 promotes loading of RAD51, enabling strand invasion of the donor duplex [3, 4].
  • HDR at nicks by single -stranded deoxyoligonucleotide (SSO) donors was stimulated 10-fold or more upon inhibition of RAD 51 [2]. Those results raised the possibility that HDR by SSO donors uses an alternative pathway distinct from canonical HDR.
  • ADSS annealing-dependent strand synthesis
  • HDR by SSO donors at nicks but not DSBs is stimulated by treatments that inhibit RAD51 loading onto DNA, including depletion of RAD51, BRCA2 or the BRCA2-associated factors SHFM1 and PALB2, expression of the BRC3 repeat region of BRCA2 which prevents BRCA2/RAD51 interaction [9], and expression of the dominant negative RAD51K133R mutant, which binds to but does not hydrolyze ATP [10, 11].
  • mutEJ mutagenic end-joining
  • the TL reporter has distinct fluorescence outputs that enable scoring of HDR as GFP+ cells, generated upon conversion of a 38 bp insert within the GFP coding sequence by a 17 bp heterologous donor sequence; and of a subset of mutEJ events as mCherry+ cells, generated by indels that cause a shift to the +2 reading frame.
  • HDR was assayed in HEK293T cells carrying the chromosomal TL reporter and treated with siBRCA2 to inhibit canonical HDR.
  • 17 nt heterologous region is flanked by 41 nt homology arms that support HDR by either the cl or cN pathway, depending on the target nick site (Fig. IB).
  • HDR occurs by the cl pathway at nicks generated by Cas9D10A using guide RNAs g2 or g8 and the SSO-2 donor, or guide RNAs gl or g9 and the SSO-1 donor (Fig. IB). With these guide RNA/donor pairs, HDR frequencies varied over a 5-fold range (0.8-4.0%; Fig. 1C).
  • HDR occurs by the cN pathway at nicks generated by Cas9D10A using guide RNAs gl or g9 and the SSO-2 donor, or guide RNAs g2 or g8 and the SSO-1 donor (Fig. IB). With these guide RNA/donor pairs, HDR frequencies varied over a 50-fold range (0.1-4.5%).
  • a 99 nt single-stranded donor SSO-3
  • SSO-3 a 99 nt single-stranded donor
  • RFPs restriction fragment polymorphisms
  • GFP+ cells were sorted, DNA isolated, and the region targeted for HDR was amplified by PCR, cleaved with Hindlll or Apol, and fragments resolved by gel electrophoresis to quantify sequence conversion.
  • the cl pathway supports use of SSO-3 at nicks targeted gl (Fig.
  • cN pathway supports use of SSO-3 at nicks targeted by g8 (Fig. 2A), and in cells corrected following targeting by g8, only the Hindlll site was transferred at an appreciable frequency (Apol 2%, Hindlll 40%; Fig. 2B).
  • sequence conversion by the cl and cN pathways indicates that these pathways are mechanistically distinct, and supports the hypothesis that cN donors use an annealing-dependent strand synthesis (ADSS) pathway distinct from the pathway used by cl donors.
  • ADSS annealing-dependent strand synthesis
  • the strategies for RAD51 inhibition included expression of the inhibitory BRC3 repeat region of BRCA2, which competes with BRCA2 for interaction with RAD51 [14]; expression of the dominant-negative RAD51K133R ATPase mutant [15]; and treatment with siRNAs targeting RAD51, BRCA2, and BRCA2-interacting factors SHFM1 and PALB2 [16]. All these approaches stimulated HDR at nicks by SSO donors from 10-fold (RAD51K133R expression) to more than 40-fold (siBRCA2 treatment; Table 1). In all cases, increased frequencies of HDR at nicks by SSO donors contrasted with reduced frequencies of HDR at either nicks or DSBs by dsDNA donors (Table 1).
  • Table 1 HDR frequencies in cells in which RAD51 is inhibited SSO donor SSO donor dsDNA donor
  • RAD51 and BRCA2 have roles in maintenance of genomic stability independent of HDR [17-21].
  • inhibition of RAD51 affected the frequency of mutEJ at nicks, the frequency of mCherry+ cells was determined in populations in which nicks were targeted by g9 in the presence of SSO-2; or gl in the presence of SSO-1.
  • mutEJ frequencies were 0.02% (Table 2).
  • Table 2 mutEJ frequencies in cells expressing or treated with inhibitors of canonical
  • RPA is required for HDR at nicks by SSO donors
  • RPA coats single -stranded DNA to enable a wide range of nuclear transactions [22, 23] .
  • canonical HDR at DSBs ssDNA ends exposed by resection are bound by RPA, then BRCA2 replaces RPA with RAD51, enabling invasion of a duplex DNA donor [4].
  • HDR frequencies were assayed in cells treated with siRPAl, which targets the largest subunit of the RPA heterotrimer.
  • siRPAl treatment reduced the frequencies of HDR at nicks, as especially evident in cells expressing BRC3 (4.7-fold; p ⁇ 0.02; Table 3).
  • siRPAl treatment also reduced frequencies of HDR at DSBs (4.3-fold; p ⁇ 0.002; Table 3), as predicted by other results [22, 24]. These results indicate that RPA bound to ssDNA stimulates HDR at nicks by SSO donors.
  • HDR at DSBs by SSO donors occurs predominately by annealing-dependent strand synthesis (ADSS)
  • SSOs can support HDR at DSBs [25] . To determine if this occurs by pathways related to those that support HDR at nicks, HDR frequencies were compared at DSBs targeted by gl, g2, g8 and g9, and supported by SSO-1 or SSO-2, or by a duplex plasmid DNA donor, pCVL SFFV dl4GFP (Fig. 3A). The SSO donors supported HDR with frequencies varying over a 15-fold range, from ⁇ 0.4% (SSO-2 at gl, g2, g8) to > 2% (SSO-1 at gl or g8; SSO-2 at g9).
  • the duplex plasmid donor supported HDR with frequencies varying over a 2.5-fold range, from 2% (g2) to 5% (g9). At any given DSB site, the duplex donor supported somewhat more efficient HDR than either SSO donor, but in no case was the duplex donor more than twice as efficient as the better SSO donor at that site.
  • ADSS annealing-dependent strand synthesis
  • SSO's represent a valuable alternative to duplex donors for genome engineering applications. They are very convenient to generate, persist for a limited time in the nucleus, and offer the potential of multiplexing targeted mutagenesis. At nicks, use of SSO donors is greatly stimulated by a variety of treatments that inhibit RAD51 activity on DNA. Under these conditions SSO donors support HDR at higher frequencies than duplex plasmid donors. At DSBs, SSOs support HDR by a pathway independent of RAD51/BRCA2, with frequencies about half those of duplex plasmid donors.
  • the ADSS pathway transfers only sequences from the 5' end of the donor to the target, and requires homology between the 3' end of the target and the SSO donor.
  • the cl pathway transfers both 5' and 3' donor sequences to the target, and is more tolerant of heterology although extensive heterology with the 5' end of the nick does limit HDR.
  • the interaction of Cas9 with its DNA target could also impact the efficiency of HDR by different SSO donors at different target sites.
  • Cas9D10A nicks the strand annealed to the guide RNA and, in vitro, remains on the nicked strand while releasing the intact strand making it accessible for annealing [26] .
  • targets nicked by Cas9D10A would be somewhat more permissive for cl than cN donor annealing, but a consistent difference between efficiencies of those donors was observed that could be explained by assymetric release of the target by Cas9D 10A.
  • dwell times of Cas9 or Cas9D10A on DNA can be influenced by helicases and chromatin remodeling activities in living cells, and rates of release of cleaved target strands may vary between cell types, or even from locus to locus within a given cell type, these data strongly indicate that the primary contribution to HDR efficiency is the relationship between nick (or DSB) site and donor heterology.
  • RAD51 may suppress genomic instability at nicks
  • RAD51 inhibits HDR by SSO donors at nicks but not DSBs.
  • a DSB undergoes 5 '-3' resection to expose 3' single-stranded tails, RPA binds these single stranded regions, activates the ATR kinase, and then is replaced by RAD51, which is loaded onto DNA by BRCA2 and its accessory factors, including SHFM1 and PALB2.
  • the RAD51-coated single -stranded 3' tails of a resected DSB (Fig. 4, left) can invade a duplex donor or anneal directly with an SSO donor.
  • a nick does not appear to undergo extensive 5'-3' resection, as suggested by the inhibitory effect of 5' heterology on the use of a cl donor (Fig. 1C).
  • the inhibitory effect of 3' heterology on the use of a cN donor similarly indicates that resection from the 3' end of a nick is limited.
  • a nick may undergo unwinding to expose a single -stranded region 3 ' and/or 5 ' of the nick on the nicked strand, and a gap on the intact.
  • Exposed single-stranded regions are probably bound by RPA, which is necessary for HDR at nicks (Table 3), which is then replaced by RAD51.
  • Treatments that inhibit RAD51 loading or activity stimulate HDR at nicks by SSO donors, including knockdown of RAD51, BRCA2, SHFM1 or PALB2; upon expression of the BRC3 peptide, which inhibits interaction of RAD51 and BRCA2; or the dominant negative RAD51K133R mutant. This indicates that RAD51 may favor reannealing of the complementary target strands, thus disfavoring HDR by SSO donors (Fig. 4, right).
  • RAD51 can also promote ligation [27,28]. Reannealing and re ligation will prevent HDR by SSO donors, and may also prevent other disadvantageous repair events at nicks, such as mutEJ, as indicated by evidence that mutEJ frequencies at nicks (but not DSBs) are elevated when RAD51 activity is reduced (Table 2). This identifies a new and unanticipated role for RAD51 in maintenance of genomic stability, and indicate a new mechanism that promotes genomic instability in tumors deficient in RAD51 or factors that promote RAD51 loading onto DNA.
  • Plasmids The pCas9D10A -T2A-BFP expression plasmid was created by swapping the Spel-Sbfl fragment of pCas9D10A [2], which contains the D10A mutation, into the Spel-Sbfl sites of pCas9(wt)-T2A-mTagBFP.
  • Donor oligonucleotide sequences Sequences of donor oligonucleotides are shown below, with regions that are homologous with the target in upper cases, and regions of heterology in lower case.
  • SSO-4 protects them or the converted target from cleavage.
  • SSO-4 which is >96% homologous with the target, was designed to carry a single nt insertion plus three sequence polymorphisms to protect both the SSO donor and the converted genomic target from cleavage, and these are shown in lowercase.
  • SSO-1 (SEQ ID NO: 14):
  • HEK 293T cells stably transduced with the Traffic Light (TL) reporter, as previously described [2] .
  • the TL reporter [13] bears a defective GFP gene in tandem with an mCherry gene in the +2 reading frame, so neither protein is correctly expressed and cells are GFP-mCherry-.
  • HDR that replaces a 38 bp insertion in the defective GFP gene with 17 nt of heterologous donor sequence will generate a functional GFP gene and GFP+ cells; and HDR that substitutes a sequence bearing a 1 nt insertion enables mCherryTM to be correctly translated, generating mCherryTM+ cells.
  • HEK293T were grown at 37°C, 5% CO 2 in Dulbecco-modified Eagle's medium (Hyclone) supplemented with 10% fetal bovine serum (Gemini Bio-Products), 200 units/ml penicillin, 200 ⁇ g/ml streptomycin (Hyclone) and 2 mM L-glutamine (Hyclone).
  • Transfections were performed using Lipofectamine RNAiMAX (Life Technologies) for siRNA and Lipofectamine LTX (Life Technologies) for plasmids and SSOs according to the manufacturer's protocol. Briefly, on day 0, 293T cells were seeded in a 96-well plate at approximately 4 x 10 3 cells per well in 100 ⁇ medium.
  • siBRCA2 or siRAD51 Thermo-Fisher ScientificTM; siRNA ID# s2085 and si 1734, respectively; a mixture of 0.125 ⁇ RNAiMAXTM, 0.5 ⁇ of 0.625 ⁇ siRNA and 9.875 ⁇ of OptiMEMTM (Life TechnologiesTM) was used to transfect each well.
  • Flow cytometry For flow cytometry analysis, cells from two wells of a 96-well plate were, washed with PBS, trypsinized, pooled, fixed in 2% formaldehyde and analyzed on an LSR II flow cytometer (Becton Dickinson). Typically 50,000 events were gated for linear side scatter and forward scatter to identify cells, and cells gated for linear side scatter height and width to eliminate doublets. In all experiments Cas9 was co-expressed with mTagBFP (BFP) to enable identification of transfectants, and data are presented as GFP+ and mCherry+ frequencies among BFP+ cells.
  • BFP mTagBFP
  • GFP, mCherry, and mTagBFP fluorescence were detected with 488 nm, 561 nm and 406 nm lasers, respectively. Data were analyzed using Flow JoTM (Tree StarTM) and frequencies were transferred to Microsoft Excel. Statistical significance was determined by two-tailed t-test.
  • Replication protein A single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair. Bioessays, 2014. 36(12): p. 1156-61.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés de fabrication d'un acide nucléique donneur simple brin destiné à être utilisé dans la modification du génome au niveau de cassures ciblées. L'invention concerne également des procédés permettant de lancer une autre modification dirigée par homologie à l'aide d'acides nucléiques donneurs simple brin tel que décrit ici pour une modification génétique ciblée efficace. Des aspects de l'invention concernent une sélection optimisée ou des cibles et la conception de l'acide nucléique donneur simple brin en vue d'obtenir une fréquence accrue de réparation dirigée par homologie (HDR) au niveau de cassures dans la cible sélectionnée pour une modification génomique efficace.
PCT/US2016/042463 2015-07-17 2016-07-15 Procédés de maximisation de l'efficacité de correction de gène cible WO2017015101A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562194049P 2015-07-17 2015-07-17
US62/194,049 2015-07-17

Publications (1)

Publication Number Publication Date
WO2017015101A1 true WO2017015101A1 (fr) 2017-01-26

Family

ID=57834494

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/042463 WO2017015101A1 (fr) 2015-07-17 2016-07-15 Procédés de maximisation de l'efficacité de correction de gène cible

Country Status (1)

Country Link
WO (1) WO2017015101A1 (fr)

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
WO2019157326A1 (fr) 2018-02-08 2019-08-15 Zymergen Inc. Édition de génome à l'aide de crispr dans corynebacterium
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
WO2020047344A1 (fr) * 2018-08-31 2020-03-05 Yale University Compositions et méthodes permettant d'améliorer l'édition génique à base d'oligonucléotides donneur
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
CN112912502A (zh) * 2018-08-31 2021-06-04 耶鲁大学 用于增强基于三链体和核酸酶的基因编辑的组合物和方法
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11850284B2 (en) 2019-08-30 2023-12-26 Yale University Compositions and methods for delivery of nucleic acids to cells
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12031126B2 (en) 2023-12-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140068797A1 (en) * 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US20140170753A1 (en) * 2012-12-12 2014-06-19 Massachusetts Institute Of Technology Crispr-cas systems and methods for altering expression of gene products
WO2014172458A1 (fr) * 2013-04-16 2014-10-23 University Of Washington Through Its Center For Commercialization Activation d'une autre voie pour la réparation dirigée par homologie afin de stimuler la correction génétique et le génie génomique
US20140357530A1 (en) * 2012-12-12 2014-12-04 The Broad Institute Inc. Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof
WO2015070083A1 (fr) * 2013-11-07 2015-05-14 Editas Medicine,Inc. Méthodes et compositions associées à crispr avec arng de régulation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140068797A1 (en) * 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US20140170753A1 (en) * 2012-12-12 2014-06-19 Massachusetts Institute Of Technology Crispr-cas systems and methods for altering expression of gene products
US20140357530A1 (en) * 2012-12-12 2014-12-04 The Broad Institute Inc. Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof
WO2014172458A1 (fr) * 2013-04-16 2014-10-23 University Of Washington Through Its Center For Commercialization Activation d'une autre voie pour la réparation dirigée par homologie afin de stimuler la correction génétique et le génie génomique
WO2015070083A1 (fr) * 2013-11-07 2015-05-14 Editas Medicine,Inc. Méthodes et compositions associées à crispr avec arng de régulation

Cited By (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
WO2019157326A1 (fr) 2018-02-08 2019-08-15 Zymergen Inc. Édition de génome à l'aide de crispr dans corynebacterium
WO2020047344A1 (fr) * 2018-08-31 2020-03-05 Yale University Compositions et méthodes permettant d'améliorer l'édition génique à base d'oligonucléotides donneur
CN112912502A (zh) * 2018-08-31 2021-06-04 耶鲁大学 用于增强基于三链体和核酸酶的基因编辑的组合物和方法
CN112930399A (zh) * 2018-08-31 2021-06-08 耶鲁大学 用于增强基于供体寡核苷酸的基因编辑的组合物和方法
JP2021534788A (ja) * 2018-08-31 2021-12-16 イェール ユニバーシティーYale University 三重鎖およびヌクレアーゼベースの遺伝子編集を亢進するための組成物および方法
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11850284B2 (en) 2019-08-30 2023-12-26 Yale University Compositions and methods for delivery of nucleic acids to cells
US11872286B2 (en) 2019-08-30 2024-01-16 Yale University Compositions and methods for delivery of nucleic acids to cells
US12005121B2 (en) 2019-08-30 2024-06-11 Yale University Compositions and methods for delivery of nucleic acids to cells
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12031126B2 (en) 2023-12-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Similar Documents

Publication Publication Date Title
WO2017015101A1 (fr) Procédés de maximisation de l'efficacité de correction de gène cible
JP7431891B2 (ja) 化学的に修飾されたガイドｒｎａを使用する高特異性ゲノム編集
US11667903B2 (en) Tracking and manipulating cellular RNA via nuclear delivery of CRISPR/CAS9
JP2021118714A (ja) Ｃｒｉｓｐｒ系組成物及び使用方法
EP3765616B1 (fr) Nouveaux systèmes et enzymes de ciblage d'adn et d'arn crispr
JP7506405B2 (ja) 真核生物の遺伝子編集のためのレンチウイルスベースのベクターならびに関連システムおよび方法
JP2023022254A (ja) Ｃｒｉｓｐｒ／ｃｐｆ１システム及び方法
US20160264934A1 (en) METHODS FOR MODULATING AND ASSAYING m6A IN STEM CELL POPULATIONS
CN109415729B (zh) 具有降低毒性的基因编辑试剂
CA3026110A1 (fr) Nouvelles enzymes crispr et systemes associes
KR20220004674A (ko) Rna를 편집하기 위한 방법 및 조성물
CA3072610A1 (fr) Methodes et compositions de ciblage d'arn
JP2020534812A (ja) 改良された相同組換えおよびその組成物のための方法
JP2024009934A (ja) 細胞ゲノムにおける、相同組換え修復（ｈｄｒ）の効率を上昇させるための方法
EP3414333A1 (fr) Système transposon réplicatif
Kroustallaki et al. SMUG1 promotes telomere maintenance through telomerase RNA processing
US20180064746A1 (en) Assay for telomere length regulators
US20230046668A1 (en) Targeted integration in mammalian sequences enhancing gene expression
CN116490615A (zh) 工程化CRISPR-Cas13f系统及其用途
Chakraborty et al. Combination of engineered FnCas9 and extended gRNAs for PAM-flexible, robust and nucleobase specific editing and diagnostics
US20230340437A1 (en) Modified nucleases
Elguindy Regulation of Pumilio RNA Binding Proteins by Long Noncoding RNA NORAD
Cherney Recruitment of PRC2 by lncRNAs
Hasler The Role of the Lupus Autoantigen La in the Human MicroRNA Pathway
Bhatt From Structure to Function: Exploring the Importance of Non-canonical Secondary Structures in RNA

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16828298

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16828298

Country of ref document: EP

Kind code of ref document: A1