Genome editing systems comprising repair-modulating enzyme molecules and methods of their use

A genome editing system comprising:

• (a) a gRNA molecule;
• (b) a RNA-guided nuclease molecule;
• and
• (c) a heterologous Repair-Modulating Enzyme Molecule (RMEM),
• wherein the gRNA molecule and the RNA-guided nuclease molecule are configured to associate with a target nucleic acid, resulting in a cleavage event;
• wherein the cleavage event is repaired by at least one DNA repair pathway that is modulated by the RMEM;
• and
• A) wherein, when the RMEM is Rad52, a frequency of the DNA repair pathway repairing the target nucleic acid:
• i) to comprise a deletion is decreased;
• ii) using gene conversion is decreased;
• and/or
• iii) using gene correction is increased
• in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid i) to comprise a deletion, ii) using gene conversion, and/or iii) using gene correction in the absence of the RMEM;
• B) wherein, when the RMEM is 53BP1 dominant negative, a frequency of the DNA repair pathway repairing the target nucleic acid:
• i) using gene conversion is increased;
• ii) using gene correction is increased;
• and/or
• iii) to comprise an insertion is decreased
• in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid i) using gene conversion, ii) using gene correction, and/or iii) to comprise an insertion in the absence of the RMEM;
• C) wherein, when the RMEM is TdT, a frequency of the DNA repair pathway repairing the target nucleic acid:
• i) using gene conversion is decreased;
• ii) using gene correction is decreased;
• iii) to comprise an insertion is increased;
• and/or
• iv) to comprise a deletion is decreased;
• in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid i) using gene conversion, ii) using gene correction, iii) to comprise an insertion, and/or iv) to comprise a deletion in the absence of the RMEM;
• D) wherein, when the RMEM is Rad51, a frequency of the DNA repair pathway repairing the target nucleic acid:
• i) using gene conversion is decreased;
• and/or
• ii) using gene correction is decreased;
• in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid i) using gene conversion and/or ii) using gene correction in the absence of the RMEM;
• E) wherein, when the RMEM is RPA, a frequency of the DNA repair pathway repairing the target nucleic acid using gene conversion is decreased in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid using gene conversion in the absence of the RMEM;
• F) wherein, when the RMEM is Artemis, a frequency of the DNA repair pathway repairing the target nucleic acid to comprise a deletion is increased in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid to comprise a deletion in the absence of the RMEM;
• G) wherein, when the RMEM is T5 exonuclease, a frequency of the DNA repair pathway repairing the target nucleic acid:
• i) using gene correction is decreased;
• and/or
• ii) to comprise an insertion is decreased;
• in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid i) using gene correction and/or ii) to comprise an insertion in the absence of the RMEM;
• or
• H) wherein, when the RMEM is ERCC1, a frequency of the DNA repair pathway repairing the target nucleic acid using gene conversion is decreased in the presence of the RMEM, as compared to a frequency of the DNA repair pathway repairing the target nucleic acid using gene conversion in the absence of the RMEM.

A method of altering a cell, comprising the steps of:

• A) forming, in a deoxyribonucleic acid (DNA) of a cell, at least one single- or double-strand break, thereby exposing at least one single-stranded DNA segment proximate to the single- or double-strand break;
• and
• annealing an exogenous single-stranded oligonucleotide donor template to the at least one single-stranded DNA segment,
• wherein (a) the annealing of the exogenous single-stranded oligonucleotide donor template is facilitated by an exogenous Rad52 protein, and (b) the single- or double-stranded break is repaired in a manner that incorporates at least a portion of a sequence of the exogenous single-stranded oligonucleotide donor template or a reverse complement thereof;
• B) forming, in a deoxyribonucleic acid (DNA) of a cell, at least one double-strand break, thereby exposing at least one single-stranded DNA segment proximate to the double-strand break;
• and
• annealing an exogenous single-stranded oligonucleotide donor template to the at least one single-stranded DNA segment,
• wherein (a) the one or more free DNA ends exposed by the double-strand break is facilitated by an exogenous 53BP1 dominant negative protein, and (b) the single- or double-stranded break is repaired in a manner that incorporates at least a portion of a sequence of the exogenous single-stranded oligonucleotide donor template or a reverse complement thereof;
• C) forming, in a deoxyribonucleic acid (DNA) of a cell, at least one single- or double-strand break, thereby generating at least one free 3′ end of the DNA;
• and
• adding at least one nucleotide to the free 3′ end of the DNA at the at least one single- or double-strand break,
• wherein (a) the single- or double-strand break is repaired in a manner that incorporates the at least one nucleotide added to the 3′ end of the DNA strand, and (b) wherein the at least one nucleotide is added by an exogenous terminal deoxynucleotidyl transferase (TdT) protein;
• D) forming, in a deoxyribonucleic acid (DNA) of a cell, at least one single- or double-strand break, thereby generating at least one free 5′ end of the DNA;
• and
• removing at least one nucleotide from the free 5′ end of the DNA at the at least one single- or double-strand break,
• wherein (a) the single- or double-strand break is repaired in a manner that deletes the at least one nucleotide from the 5′ end of the DNA strand, and (b) wherein the at least one nucleotide is deleted by an exogenous Artemis protein;
• or
• E) forming, in a deoxyribonucleic acid (DNA) of a cell, at least one single- or double-strand break, thereby generating a 3′ single-stranded overhang and/or a 5′ single-stranded overhang in the DNA;
• and
• cleaving a phosphodiester backbone of the DNA to remove at least a portion of the 3′ single-stranded overhang and/or the 5′ single-stranded overhang,
• wherein (a) the phosphodiester backbone of the DNA is cleaved by an exogenous ERCC1 protein, and (b) wherein the single- or double-stranded break is repaired in a manner that inhibits incorporation of at least a portion of a sequence of an exogenous single-stranded oligonucleotide donor template, or reverse complement thereof.