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… and methods for improving homogeneity of dna generated using a crispr/cas9 … — Christiana Care Gene Editing Institute, LLC (US20230091847A1)

Christiana Care Gene Editing Institute, LLC · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US20230091847A1, Christiana Care Gene Editing Institute, LLC, Eric B. Kmiec, en, 2023

ABSTRACT

Abstract

The invention relates to the unexpected discovery of a system and methods for precise homology directed repair after CRISPR/Cas9 cleavage. The invention includes a DNA cleavage and repair system comprising a CRISPR/Cas9 system and an oligonucleotide 100% complementary to cleaved DNA to promote homology directed DNA repair. The invention further includes methods for inducing homology directed repair of cleaved DNA and repairing a CRISPR/Cas9 cleavage.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser. No. 15/402,833, filed Jan. 10, 2017, which claims priority under 35 U.S.C. Â§ 119(e) to U.S. Provisional Patent Application No. 62/277,212, filed Jan. 11, 2016, and U.S. Provisional Patent Application No. 62/442,145, filed Jan. 4, 2017, all of which are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM109021-02 awarded by National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The Sequence Listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 130949-00402_SL.xml. The size of the text file is 55 KB, and the text file was created on Oct. 27, 2022.

BACKGROUND OF THE INVENTION

Knocking genes out (also known as âgene knockoutsâ or âgene knock-outsâ) using the CRISPR/Cas9 system has proven much easier than knocking genes in (also known as âgene knockinsâ or âgene knock-insâ) using that system, because double-stranded breaks induced by the Cas9 nuclease are more quickly repaired by the non-homologous end-joining (NHEJ) DNA repair mechanism. It should be noted that NHEJ often drops nucleotides from the ends being joined. This works well for inducing gene knock-outs (because it leads to a frameshift), but is not ideal for gene knock-ins (which requires greater precision).

There is thus a need to promote precise gene editing using the CRISPR/Cas9 systems. Unfortunately, the homology-directed repair (HDR) pathway, which allows for insertion of precise genetic modifications, has low efficiency compared with the NHEJ pathway. Eliminating Ku heterodimer proteins and DNA ligase IV involved in the NHEJ pathway increases efficiency of HDR. However, the most effective way of inhibiting the error-prone NHEJ pathway is to degrade these molecules with adenovirus 4 proteins. This approach relies on specific or targeted degradation of native DNA repair machinery, and does not provide an optimal approach for gene therapy.

Therefore, a need exists in the art for efficient and precise gene editing methods, which can be used for example in gene therapy. Such methods should allow gene knock-ins using CRISPR/Cas9 technologies without frameshift. The present invention satisfies this need.

BRIEF SUMMARY OF THE INVENTION

As described herein, the present invention relates in part to compositions and methods for promoting precise gene editing (such as, but not limited to, âknock-inâ and/or âknock-outâ) using CRISPR/Cas9 systems.

In one aspect, the invention includes a method for promoting DNA cleavage and repair in a cell. In another aspect, the invention includes a method for inducing homology directed repair of a DNA cleaved by a CRISPR/Cas9 system in a cell.

In certain embodiments, the method utilizes at least one CRISPR/Cas9 system and a deoxyoligonucleotide. The at least one CRISPR/Cas9 system cleaves in a cell a DNA in one or more sites, and generates a first cleavage end and a second cleavage end on the DNA. The deoxyoligonucleotide comprises two regions, wherein the first region is complementary to the first generated cleavage end and at least one deoxynucleotide adjacent to the first generated cleavage end of the DNA, and wherein the second region is complementary to the second generated cleavage end and at least one deoxynucleotide adjacent to the second generated cleavage end of the DNA. In certain embodiments, the first region of the deoxyoligonucleotide anneals to the first generated cleavage end and the at least one deoxynucleotide adjacent to the first cleavage end of the DNA, and the second region of the deoxyoligonucleotide anneals to the second generated cleavage end and the at least one deoxynucleotide adjacent to the second cleavage end of the DNA. In certain embodiments, the DNA is cleaved and repaired with no insertion or deletion of one or more unwanted deoxynucleotides at the junction of the first and second cleavage ends of the DNA.

In yet another aspect, the invention includes a method of promoting fusion, without insertion or deletion of unwanted deoxynucleotides, of first and second cleavage ends of a DNA that was cleaved by a CRISPR/Cas9 system in a cell. The method comprises providing a DNA that has been cleaved in one or more sites by at least one CRISPR/Cas9 system in the cell, wherein the cleaved DNA comprises a first cleavage end and a second cleavage end. The method further comprises annealing to the cleaved DNA a deoxyoligonucleotide comprising two regions, wherein the first region is complementary to the first generated cleavage end and at least one deoxynucleotide adjacent to the first generated cleavage end of the DNA, and wherein the second region is complementary to the second generated cleavage end and at least one deoxynucleotide adjacent to the second generated cleavage end of the DNA. In certain embodiments, homology directed repair is induced at the generated cleavage ends with no insertion or deletion of one or more unwanted deoxynucleotides at the junction of the first and second cleavage ends.

In one aspect, the invention includes a method for promoting DNA fusing and repair in a cell. In another aspect, the invention includes a method for inducing homology directed fusing of a first DNA cleaved by a CRISPR/Cas9 system in a cell with a second DNA.

In certain embodiments, the method utilizes at least one CRISPR/Cas9 system and a deoxyoligonucleotide. The at least one CRISPR/Cas9 system cleaves in a cell a first DNA in one or more sites, and generates a first cleavage end on the DNA. A second DNA, which is to be fused to the first DNA, comprises at least a second cleavage end. The deoxyoligonucleotide comprises two regions, wherein the first region is complementary to the first generated cleavage end and at least one deoxynucleotide adjacent to the first generated cleavage end of the first DNA, and wherein the second region is complementary to the second cleavage end and at least one deoxynucleotide adjacent to the second cleavage end of the second DNA. In certain embodiments, the first region of the deoxyoligonucleotide anneals to the first generated cleavage end and the at least one deoxynucleotide adjacent to the first cleavage end of the first DNA, and the second region of the deoxyoligonucleotide anneals to the second cleavage end and the at least one deoxynucleotide adjacent to the second cleavage end of the second DNA. In other embodiments, the first DNA is fused to the second DNA with no insertion or deletion of one or more unwanted deoxynucleotides at the junction of the first cleavage end of the first DNA and the second cleavage end of the second DNA.

In yet another aspect, the invention includes a method of promoting fusion, without insertion or deletion of unwanted deoxynucleotides, of a first cleavage end of a first DNA that was cleaved by a CRISPR/Cas9 system in a cell with a second cleavage end of a second DNA. The method comprises providing a first DNA that has been cleaved at a first cleavage site by at least one CRISPR/Cas9 system in the cell, and a second DNA comprising a second cleavage site. The method further comprises annealing to the first and second DNAs a deoxyoligonucleotide comprising two regions, wherein the first region is complementary to the first generated cleavage end and at least one deoxynucleotide adjacent to the first generated cleavage end of the first DNA, and wherein the second region is complementary to the second cleavage end and at least one deoxynucleotide adjacent to the second cleavage end of the second DNA. In certain embodiments, fusing with homology directed repair of the first and second DNAs is promoted, with no insertion or deletion of one or more unwanted deoxynucleotides at the junction of the first cleavage end of the first DNA and the second cleavage end of the second DNA.

In yet another aspect, the invention provides a kit for promoting DNA cleavage and repair in a cell. In yet another aspect, the invention provides a kit for inducing homology directed repair of a DNA cleaved by a CRISPR/Cas9 system in a cell.

In certain embodiments, the kit comprises at least one CRISPR/Cas9 system, which cleaves in a cell a DNA in one or more sites, and generates a first cleavage end and a second cleavage end on the DNA. In other embodiments, the kit further comprises a deoxyoligonucleotide comprising two regions, wherein the first region is complementary to the first generated cleavage end and at least one deoxynucleotide adjacent to the first generated cleavage end of the DNA, and wherein the second region is complementary to the second generated cleavage end and at least one deoxynucleotide adjacent to the second generated cleavage end of the DNA.

In yet another aspect, the invention provides a kit for promoting DNA fusing and repair in a cell. In yet another aspect, the invention provides a kit for inducing homology directed fusing of a first DNA cleaved by a CRISPR/Cas9 system in a cell with a second DNA.

In certain embodiments, the kit comprises at least one CRISPR/Cas9 system, which cleaves in a cell a DNA in one or more sites, and generates a first cleavage end on the DNA. In other embodiments, the kit further comprises a second DNA comprising a second cleavage end. In yet other embodiments, the kit further comprises a deoxyoligonucleotide comprising two regions, wherein the first region is complementary to the first generated cleavage end and at least one deoxynucleotide adjacent to the first generated cleavage end of the first DNA, and wherein the second region is complementary to the second cleavage end and at least one deoxynucleotide adjacent to the second cleavage end of the second DNA.

In various embodiments of the above aspects or any other aspect of the invention delineated herein, the deoxyoligonucleotide comprises about 25 to about 200 deoxynucleotides in length. In other embodiments, the one or more unwanted insertion or deletion comprises a single deoxynucleotide. In yet other embodiments, the one or more unwanted insertion or deletion comprises more than one deoxynucleotide. In yet other embodiments, the at least one deoxynucleotide adjacent to the first generated cleavage end comprises about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 and/or 100 deoxynucleotides. In yet other embodiments, the at least one deoxynucleotide adjacent to the second generated cleavage end comprises about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 and/or 100 deoxynucleotides. In yet other embodiments, the first region is directly linked to the second region. In yet other embodiments, the first region is linked to the second region through an oligonucleotide comprising about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or any fraction or multiple thereof, deoxynucleotides. In yet other embodiments, the CRISPR/Cas9 system is derived from a plasmid.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

FIG. 1 A is a schematic diagram illustrating an experimental workflow of certain methods described herein. HCT116-19 cells are either unsynchronized or synchronized and released, then transfected with a CRISPR/Cas9 expression vector (pX330) with or without a single stranded oligodeoxynucleotide (ssODN). After 48 hours, the cells are analyzed for gene editing activity, Surveyor endonuclease digestion, and RFLP.

FIG. 1 B illustrates the sequence of a ssODN used in a gene editing system (SEQ ID NO: 1) aligned with the wild-type (SEQ ID NO: 2) and mutant eGFP gene (SEQ ID NO: 3). The wild-type and mutated eGFP gene segments with the target codon located in the center of the sequences are shown. The nucleotide targeted for exchange is emphasized in bold and underlined. Phosphorothioate modified and end protected (denoted with *) 72NT, a 72-mer, used to target the non-transcribed (NT) strand of the mutated eGFP gene is shown.

FIG. 2 is a graph illustrating a gene editing dose curve using synchronized and unsynchronized cells. Synchronized (black) and unsynchronized (grey) HCT116-19 cells were electroporated with 0.1-10.0 Î¼g of pX330 and 1.35 Î¼g of 72NT. After a 48-hour recovery period, gene editing activity was measured using a GUAVA EASYCYTE 5HTÂ® (Millipore) multiparameter flow cytometer. Gene editing is displayed as correction efficiency (%), determined by the number of viable eGFP positive cells, divided by the total number of viable cells in the population. Each treatment was performed in triplicate and standard error is illustrated with accompanying bars.

FIG. 3 is a graph illustrating CRISPR/Cas9 activity as measured by SURVEYOR MUTATION DETECTIONÂ® kits (Integrated DNA Technologies) and restriction fragment length polymorphism analysis (RFLP) vs gene editing as measured by FACS. Synchronized and released HCT116-19 cells were electroporated with 0.0-10.0 Î¼g of pX330 and with (+ODN) or without (âODN) 1.35 Î¼g of 72NT.

FIG. 4 A is a panel of graphs illustrating CRISPR effects on cell cycle progression. HCT116-19 cells were synchronized with 6 Î¼M aphidicolin for 24 hours and released for an additional 4 hours in culturing medium. Synchronized and unsynchronized HCT116-19 cells were simultaneously transfected at a concentration of 5Ã10 5 cells/with 1.35 Î¼g single-stranded oligonucleotide and 3 Î¼g CRISPR/Cas9 plasmid constructs. Cells were allowed to recover in complete growth media for 24 hr. Cell cycle profiles illustrate DNA content distributions of the cells at 24 hr post transfection. Cell cycle modeling was performed using the auto analysis feature of the MODFIT LTâ¢ software (Verity Software House). S-phase extension was by Diploid (%), Diploid: S-phase (%), and Total S-Phase (%) (Average S-Phase). Debris (%) was also analyzed to determine the quality of the analyzed data.

FIG. 4 B is a table illustrating percentages of cells present in the cell cycle stages measured in FIG. 4 A .

FIG. 5 illustrates a model system for gene editing of the mutant eGFP gene. The appropriate segments of the wild-type and mutated eGFP gene with the targeted codon, located in the center of the sequence, are displayed. The nucleotide targeted for exchange is bolded and underlined. The oligonucleotide used in these experiments is 72 bases in length bearing phosphorothioate modified linkages at the three terminal bases; the 72-mer targets the non-transcribed (NT) strand (72NT).

FIG. 6 A illustrates CRISPR/Cas9 Ribonucleoprotein Assembly Reaction. crRNA provides target specifici

CROSS-REFERENCE TO RELATED APPLICATIONS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM109021-02 awarded by National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

BACKGROUND OF THE INVENTION

BRIEF SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 4 B is a table illustrating percentages of cells present in the cell cycle stages measured in FIG. 4 A .

FIG. 6 A illustrates CRISPR/Cas9 Ribonucleoprotein Assembly Reaction. crRNA provides target specificity (20 bases) corresponding to the 2C protospacer sequence and an interaction domain (blue) with the tracrRNA. crRNA and tracrRNA are annealed in equimolar concentrations. Cas9 protein (gray) is added to complete RNP assembly. Guide RNAs (gRNAs) direct and activate the Cas9 endonuclease which then cleaves the target DNA. The lower section of the figure shows the 2C seed sequence and the tracrRNA sequence.

FIG. 6 B illustrates in vitro RNP Digestion. Genomic DNA was isolated from untreated HCT 116-19 cells and PCR used to generate an amplicon of size 605 bp, which surrounds the sequence of the integrated mutant eGFP gene. The amplicon was combined with 25 pmols and 50 pmols of RNP complex respectively, and incubated for 40 minutes at 37Â° C. In the complete reaction, two products were generated with sizes consistent with fragments predicted from the specific cut site designed for the RNP complex. As a control, the RNP complex was incubated with an amplicon generated from the HBB gene 345 base pairs in length from cell line K562. A control digest was performed on the 345 base amplicon with the restriction enzyme DdeI

FIG. 7 A illustrates the finding that gene editing is dose dependent when directed by the RNP and the ssODN. Synchronized and released HCT 116-19 cells were electroporated with 24-120 pmol CRISPR/Cas9 RNP and 0.6-3.0 Î¼M of 72 mer. After a 72-hour recovery period, gene editing activity was measured using a FACSAria II flow cytometer. Gene editing is displayed as correction efficiency (%), determined by the number of viable eGFP positive cells divided by the total number of viable cells in the population. Each treatment was performed in triplicate and standard error is illustrated with accompanying bars. Inset: Single agent gene editing. Gene editing activity directed by the single-stranded oligonucleotide (72NT) in the absence of the RNP complex under identical conditions is presented as a function of increasing concentration.

FIG. 7 B illustrates the finding that gene editing activity is dependent on all components being present in the reaction mixture. Synchronized and released HCT 116-19 cells were electroporated with 100 pmol of the crRNA, Cas9 Protein, tracrRNA and 2.0 Î¼M of the 72NT, as a complete reaction. Identical mixtures, lacking the indicated reaction component, were carried out in parallel. In one specific reaction mixture, the RNP specific for the beta globin gene replaced the RNP specific for the eGFP gene (far right bar). After a 72 hour recovery period, gene editing activity was measured using a FACSAria II flow cytometer. Gene editing is displayed as correction efficiency (%), determined by the number of viable eGFP positive cells divided by the total number of viable cells in the population. Each treatment was performed in triplicate and standard error is illustrated with accompanying bars.

FIG. 8 A illustrates FACSAria II plots of gene editing activity in HCT 116-19 cells. HCT 116-19 cells synchronized for 24 hours at the G1/S border and released were electroporated with 100 pmol of RNP complex and 2.0 Î¼M of the 72NT ssODN. After 72 hours, the cells were analyzed using FACS and single cells were sorted individually into 96-well plates. Two distinct populations were collected. The population of live, eGFP-positive cells (labeled as P2 on the FACS plot) as well as the population of live, eGFP-negative cells (labeled as P3) were segregated into separate clonal expansion plates.

FIG. 8 B illustrates experimental strategy isolation of single cell clones. Cells exhibiting eGFP expression were scored positive and sorted using a FACSAria II flow cytometer as single cells into individual wells for clonal expansion. Cells lacking eGFP expression isolated and sorted in a similar fashion and expanded under the same conditions. DNA was then isolated and the eGFP gene was amplified and subjected to Sanger sequencing to analyze gene editing activity surrounding the target site.

FIG. 9 A illustrates allelic analysis of eGFP positive cells expanded as a clonal population. Clonally isolated and expanded eGFP positive samples (sixteen clones) were analyzed at the site surrounding the targeted base and DNA from each, harvested, purified, amplified and sequenced. Allelic analysis was carried out using Sanger sequencing, assembled using SnapGene and compared to the sequence of a wild-type allele which is illustrated at the top of the figure; the cut site of the RNP complex is indicated as a small black arrow (2C crRNA).

FIGS. 9 B and 9 C illustrate allelic analysis of eGFP negative cells expanded as a clonal population. Fifteen individual samples, expanded from cloned originating from the uncorrected population were randomly selected and analyzed for indel formation at the site surrounding the target nucleotide. As above, allelic analysis was carried out using Sanger sequencing and assembled SnapGene. Once again, the sequence of a wild-type allele at the top of the figure along with the cut site of the RNP is presented.

FIG. 9 D illustrates allelic analysis of eGFP negative cells presenting insertions. Two individual clones from the uncorrected population displayed insertions of 15 bp (top panel) and 24 bp (bottom panel), respectively. The center panel represents the mutant eGFP gene sequence with the mutant codon depicted. The inserted bases are depicted with the corrected tyrosine codon depicted in light grey and the mutant stop codon represented by a red asterisk. The boundaries of the insertions are denoted by black bars.

FIG. 10 illustrates a model for point mutation repair directed by an RNP complex and a short single-stranded DNA oligonucleotide. Panels A and B: the RNP particle induces a double strand break at the target site generating two free 3â² hydroxyl ends on each strand of the broken DNA. Panel C: the oligonucleotide aligns in imperfect homologous register with the non-transcribed strand of the chromosome. The DNA replication machinery fills the gap starting from the 3â²-hydroxyl end and completing by ligation to the 5â²-phosphate at the opposite side of the gap. The single-stranded oligonucleotide serves as a template for the replication process. Panels D/E: dissociation of the single-stranded oligonucleotide allows for the newly synthesized DNA to act as a template for DNA replication in the opposite direction on the bottom strand followed by ligation.

FIG. 11 is a graph showing the distribution of insertions or deletions created by the CRISPR/Cas9 complex in the absence or presence of a single-stranded oligonucleotide. Data was compiled from 108 mammalian cell clonal expansions randomly chosen from experiments involving CRISPR activity on the eGFP gene or the human beta globin gene.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, specific materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used herein, the articles âaâ and âanâ are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, âan elementâ means one element or more than one element.

As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term âaboutâ is meant to encompass variations of Â±20% or Â±10%, more specifically Â±5%, even more specifically Â±1%, and still more specifically Â±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein the term âamountâ refers to the abundance or quantity of a constituent in a mixture.

As used herein, the term âampliconâ or âPCR productsâ or âPCR fragmentsâ or âamplificationâ products refers to extension products that comprise the primer and the newly synthesized copies of the target sequences.

As used herein, the term âbpâ refers to base pair.

The term âcomplementaryâ refers to the degree of anti-parallel alignment between two nucleic acid strands. Complete complementarity requires that each nucleotide be across from its opposite. No complementarity requires that each nucleotide is not across from its opposite. The degree of complementarity determines the stability of the sequences to be together or anneal/hybridize. Furthermore various DNA repair functions as well as regulatory functions are based on base pair complementarity.

The term âconcentrationâ refers to the abundance of a constituent divided by the total volume of a mixture. The term concentration can be applied to any kind of chemical mixture, but most frequently it refers to solutes and solvents in solutions.

The term âCRISPR/Casâ or âclustered regularly interspaced short palindromic repeats systemâ or âCRISPRâ interchangeably refers to DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of spacer DNA from previous exposures to a virus or plasmid. Bacteria and archaea have evolved adaptive immune defenses termed CRISPR/CRISPR-associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA via. RNA-guided DNA cleavage. To direct Cas9 to cleave sequences of interest, crRNA-tracrRNA fusion transcripts, hereafter referred to as âguide RNAsâ or âgRNAsâ may be designed, from human U6 polymerase III promoter. CRISPR/CAS mediated genome editing and regulation, highlighted its transformative potential for basic science, cellular engineering and therapeutics. In the type II CRISPR/Cas system, short segments of foreign DNA, termed âspacersâ are integrated within the CRISPR genomic loci and transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNAs (tracrRNAs) and direct sequence-specific cleavage and silencing of invading DNA by Cas proteins. Recent work has shown that target recognition by the Cas9 protein requires a âseedâ sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA-binding region.

The term âdownregulationâ as used herein refers to the decrease or elimination of gene expression of one or more genes.

âEffective amountâ or âtherapeutically effective amountâ are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

âEncodingâ refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein âendogenousâ refers to any material from or produced inside an organism, cell, tissue or system.

As used herein, the term âexogenousâ refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term âexpressionâ as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter

âExpression vectorâ refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

âHomologousâ as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

âIdentityâ as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

As used herein, an âinstructional materialâ includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

As used herein, âisolatedâ means altered or removed from the natural state through the actions, directly or indirectly, of a human being. For example, a nucleic acid or a peptide naturally present in a living animal is not âisolated,â but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is âisolated.â An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

The term âmeasuringâ according to the present invention relates to determining the amount or concentration, preferably semi-quantitatively or quantitatively. Measuring can be done directly and/or indirectly.

By the term âmodifiedâ as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

By the term âmodulating,â as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

A âmutationâ as used therein is a change in a DNA sequence resulting in an alteration from a given reference sequence (which may be, for example, an earlier collected DNA sample from the same subject). The mutation can comprise deletion and/or insertion and/or duplication and/or substitution of at least one deoxyribonucleic acid base such as a purine (adenine and/or thymine) and/or a pyrimidine (guanine and/or cytosine). Mutations may or may not produce discernible changes in the observable characteristics (phenotype) of an organism (subject).

By ânucleic acidâ is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. âAâ refers to adenosine, âCâ refers to cytosine, âGâ refers to guanosine, âTâ refers to thymidine, and âUâ refers to uridine.

Unless otherwise specified, a ânucleotide sequence encoding an amino acid sequenceâ includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

As used herein, one skilled in the art âobtainsâ an experimental result, data set, material, conclusion or any other piece of knowledge when one comes into possession of such experimental result, data set, material, conclusion or any other piece of knowledge, which may have been acquired by one or more third parties or by the one skilled in the art in its entirety or at least partially. In certain embodiments, one skilled in the art obtains experimental data, which may be raw data or at least partially processed data, and processes and/or manipulates the data as to reach at least one scientific conclusion or inference. In other embodiments, one skilled in the art obtains at least one scientific conclusion or inference that is derived from experimental data by one or more third parties' processing and/or manipulation. In other embodiments, one skilled in the art obtains at least one material that is identified and/or prepared by one or more third parties.

The term âoligonucleotideâ typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which âUâ replaces âTâ.

As used herein, the terms âpeptide,â âpolypeptide,â and âproteinâ are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. âPolypeptidesâ include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The term âpolynucleotideâ includes cDNA, RNA, DNA/RNA hybrid, anti-sense RNA, siRNA, miRNA, snoRNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatized, synthetic, or semisynthetic nucleotide bases. Also, included within the scope of the invention are alterations of a wild type or synthetic gene, including but not limited to deletion, insertion, substitution of one or more nucleotides, or fusion to other polynucleotide sequences.

Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5â²-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5â²-direction.

A âprimerâ is an oligonucleotide, usually of about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length, that is capable of hybridizing in a sequence specific fashion to the target sequence and being extended during the PCR.

The term âpromoterâ as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

As used herein, the terms âreferenceâ or âcontrolâ are used interchangeably, and refer to a value that is used as a standard of comparison.

The term âRNAâ as used herein is defined as ribonucleic acid.

A âsampleâ or âbiological sampleâ as used herein means a biological material from a subject, including but is not limited to organ, tissue, exosome, blood, plasma, saliva, urine and other body fluid. A sample can be any source of material obtained from a subject.

A âsingle nucleotide polymorphismâ (SNP), as referred herein, represents a variation in one or more single nucleotide changes in a DNA sequence among organisms, such among viruses, among mammals, or among humans. For instance, a SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) in a certain stretch of DNA. SNPs are the most common type of genetic variation among people and occur normally throughout a person's DNA (around 10 million SNPs in the human genome). Most commonly, these variations are found in the non-coding DNA between genes. They can act as biological markers and can be associated with certain diseases particularly when they occur within a gene or in a regulatory region near a gene. In the cases where SNPs occur within a gene, they may lead to variations in the amino acid sequence. SNPs can help predicting an individual's response to certain drugs, susceptibility to environmental factors such as toxins, and risk of developing particular diseases.

A âsubjectâ or âpatientâ as used therein may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

The term âtherapeuticâ as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

As used herein, to âtreatâ means reducing the frequency with which symptoms of a disease, disorder, or adverse condition, and the like, are experienced by a subject.

The term âtreatmentâ as used within the context of the present invention is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for the disease or disorder. Thus, for example, the term treatment includes the administration of an agent prior to or following the onset of a disease or disorder thereby preventing or removing all signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the disease comprises âtreatmentâ of the disease.

A âvectorâ is a composition of matter comprising an isolated nucleic acid, and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term âvectorâ includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

DESCRIPTION

The invention relates in one aspect to the unexpected discovery of a system and methods for precise homology directed repair (HDR) after CRISPR/Cas9 cleavage. The invention includes a DNA cleavage and repair method comprising a CRISPR/Cas9 system that cleaves DNA generating cleavage ends, and an oligonucleotide that is complementary to each of the generated cleavage ends and nucleotides adjacent to the cleavage ends. In certain embodiments, the oligonucleotide is capable of annealing to the generated cleavage ends and adjacent nucleotides with no unwanted insertion or deletion to the cleaved DNA. The invention further includes methods for inducing homology directed repair of cleaved DNA and repairing a CRISPR/Cas9 cleavage.

CRISPR/Cas

The CRISPR/Cas system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a âseedâ sequence within the guide RNA (gRNA) and a conserved tri-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR/CAS system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA for use in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR/CAS system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes.

One example of a CRISPR/Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Publication No. US2014/0068797, which is incorporated herein by reference in its entirety. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.

CRISPR/Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof.

In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

The guide nucleic acid sequence is specific for a gene and targets that gene for Cas endonuclease-induced double strand breaks. The sequence of the guide nucleic acid sequence may be within a loci of the gene. In one embodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

The guide nucleic acid sequence may be specific for any gene, such as a gene that would reduce immunogenicity or reduce sensitivity to an immunosuppressive microenvironment. The guide nucleic acid sequence includes a RNA sequence, a DNA sequence, a combination thereof (a RNA-DNA combination sequence), or a sequence with synthetic nucleotides. The guide nucleic acid sequence can be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA.

In the context of formation of a CRISPR complex, âtarget sequenceâ refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional. In certain embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In other embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulat

CLAIMS

Claims ( 10 )

1 .- 12 . (canceled)

13 . A method of exchanging a single nucleotide in a DNA sequence using a CRISPR/Cas9 system in a synchronized and released population of cells in vitro or ex vivo, the method comprising:

cleaving a first DNA within 20 bases of a single nucleotide target site in the synchronized and released population of cells using at least one CRISPR/Cas9 system comprising a ribonucleoprotein (RNP) complex, thus generating a first cleaved DNA comprising a first cleavage end; exchanging a single nucleotide at the single nucleotide target site by (a) annealing the first cleaved DNA with a single-stranded deoxyoligonucleotide about 72 nucleotides in length comprising (i) a first region 100% complementary to the first cleavage end and at least one deoxynucleotide adjacent to the first cleavage end of the first cleaved DNA, (ii) a second region 100% complementary to a second cleavage end of a second DNA and at least one deoxynucleotide adjacent to the second cleavage end of the second cleaved DNA, and (iii) a single mismatched nucleotide as compared to the single nucleotide target site of the first DNA and (b) fusing the first and second cleavage ends of the cleaved DNA with no insertion and deletion of one or more unwanted deoxynucleotides at the junction of the first cleavage end of the cleaved first DNA and the second cleavage end of the second DNA to generate a repaired DNA at the single nucleotide target site comprising a repaired nucleotide complementary to the single mismatched nucleotide of the single-stranded deoxyoligonucleotide.

14 .- 16 . (canceled)

17 . The method of claim 13 , wherein the first region of the single-stranded deoxyoligonucleotide is directly linked to the second region of the single-stranded deoxyoligonucleotide.

18 . The method of claim 13 , wherein no collateral on-site or off-site DNA mutagenesis is produced.

19 . A method of exchanging a single nucleotide in a first DNA cleaved by at least one CRISPR/Cas9 system a synchronized and released population of cells in vitro or ex vivo and a second cleavage end of a second DNA, the method comprising:

exchanging a single nucleotide at a single nucleotide target site that is within 20 bases of the CRISPR/Cas9 cleavage site without introducing an insertion or deletion of one or more unwanted deoxynucleotides by (a) annealing a single-stranded oligonucleotide about 72 nucleotides in length to the first DNA cleaved in one or more sites using at least one CRISPR/Cas9 system comprising a ribonucleoprotein (RNP) complex in the synchronized and released population of cells and (b) fusing a first cleavage end of the first DNA with the second cleavage end of the second DNA with no insertion and deletion of one or more unwanted deoxynucleotides at the junction of the first cleavage end of the first DNA and the second cleavage end of the second DNA to generate a repaired DNA comprising a repaired nucleotide complementary to the single mismatched nucleotide of the single-stranded deoxyoligonucleotide, wherein the single-stranded oligonucleotide comprises (i) a first region 100% complementary to the first cleavage end of the first DNA and at least one deoxynucleotide adjacent to the first cleavage end of the first DNA, (ii) a second region 100% complementary to the second cleavage end of the second DNA and at least one deoxynucleotide adjacent to the second cleavage end of the second DNA, and (iii) a single mismatched nucleotide as compared to the single nucleotide target site of the DNA.

20 .- 22 . (canceled)

23 . The method of claim 19 , wherein the first region of the single-stranded deoxyoligonucleotide is directly linked to the second region of the single-stranded deoxyoligonucleotide.

24 . The method of claim 19 , wherein no collateral on-site or off-site DNA mutagenesis is produced.

25 .- 26 . (canceled)

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