Methods and compositions for enhancing nuclease-mediated gene disruption
First Claim
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1. A method for targeted genomic disruption via microhomology-mediated end joining (MMEJ) in a cell, the method comprising:
- administering at least one nuclease to the cell, wherein the nuclease cleaves endogenous genomic sequences in the cell, wherein the nuclease is a zinc finger nuclease, a TALE effector domain nuclease (TALEN) and/or a CRISPR/Cas nuclease system; and
growing the cell in a medium comprising at least one small molecule inhibitor of a DNA-dependent-protein kinase catalytic subunit (DNA-PKcs) protein and a small molecule inhibitor of a Poly-(ADP-ribose) polymerase 1/2 (PARP1/2) protein at 0.5 to 25 wherein the small molecule inhibitor is a nicotinamide;
a isoquinolinone and a dihydroisoquinolinones;
a benzimidazole;
an indole;
phthalazin-1(2H)-one;
a quinazolinone;
an isoindolinone and analogues and derivatives thereof;
a phenanthridine;
a phenanthridinone;
a benzopyrone and analogues and derivatives thereof;
an unsaturated hydroximic acid derivative and analogues and derivatives thereof;
a pyridazine;
caffeine, theophylline;
thymidine and/or NU7026 and/or NU7441, wherein the endogenous genomic sequences in the cell are disrupted via MMEJ after cleavage by the at least one nuclease.
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Abstract
Methods and compositions for increasing nuclease-mediated genomic modification using DNA repair inhibitors are provided.
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8 Claims
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1. A method for targeted genomic disruption via microhomology-mediated end joining (MMEJ) in a cell, the method comprising:
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administering at least one nuclease to the cell, wherein the nuclease cleaves endogenous genomic sequences in the cell, wherein the nuclease is a zinc finger nuclease, a TALE effector domain nuclease (TALEN) and/or a CRISPR/Cas nuclease system; and growing the cell in a medium comprising at least one small molecule inhibitor of a DNA-dependent-protein kinase catalytic subunit (DNA-PKcs) protein and a small molecule inhibitor of a Poly-(ADP-ribose) polymerase 1/2 (PARP1/2) protein at 0.5 to 25 wherein the small molecule inhibitor is a nicotinamide;
a isoquinolinone and a dihydroisoquinolinones;
a benzimidazole;
an indole;
phthalazin-1(2H)-one;
a quinazolinone;
an isoindolinone and analogues and derivatives thereof;
a phenanthridine;
a phenanthridinone;
a benzopyrone and analogues and derivatives thereof;
an unsaturated hydroximic acid derivative and analogues and derivatives thereof;
a pyridazine;
caffeine, theophylline;
thymidine and/or NU7026 and/or NU7441, wherein the endogenous genomic sequences in the cell are disrupted via MMEJ after cleavage by the at least one nuclease. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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Specification