Use of Inhibitors to Increase Efficiency of CRISPR/Cas Insertions

A method of inserting a polynucleotide of interest into a genome of a eukaryotic cell, the method comprising:

• a. adding an inhibitor of the microhomology-mediated end joining (MMEJ) pathway to a composition comprising the eukaryotic cell,
• b. adding the polynucleotide of interest to the composition,
• wherein the genome comprises a genomically integrated Cas polynucleotide, and
• wherein the polynucleotide of interest is inserted into the genome by homology directed repair (HDR) or single-stranded template repair (SSTR),
• wherein the inhibitor of the MMEJ pathway is an inhibitor of PolQ, selected from a compound of formula (I):
• or any stereoisomer thereof or pharmaceutically acceptable salt thereof;
• wherein,
• R1 and R2 are each, independently, H, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 hydroxyalkyl, —CN, C2-C4 alkyne, or C2-C6 alkoxyalkyl;
• Q1, Q2, and Q3 are, independently N, C-L-R, or CRx, wherein no more than one of Q1, Q2, and Q3 is C-L-R;
• L is a bond, —O—;
• —C(O)—;
• —O(CH2)pC(O)—;
• —C(O)NRy—;
• —O(CH2)pC(O)NRy—;
• —O(CH2)pNRy;
• —NRy—;
• —(CH2)p—;
• —(CH2)pNRy—;
• —(CH2)pO—;
• —(CH2)pC(O)—;
• —(CH2)pC(O)O—;
• —O(CH2)p—;
• p is, independently, 1, 2, or 3
• R is H, Ra, Rb, Rc, or Rd;
• Ra is a 3-10 membered heterocycle optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, —S(O)2OH, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycle, and C1-C7 alkyl, wherein the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, C2-C8 ester, and C1-C5 alkoxy;
• Rb is a C1-C7 alkyl, wherein one or two methylene groups from the C1-C7 alkyl are optionally replaced with NRe or O and one or two single bonds in a C2-C7 alkyl chain are optionally replaced with a double or triple bond(s), wherein the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from:
• halo, oxo, hydroxy, carboxyl, amino, —CN, C2-C4 alkynyl, C2-C6 carbamate, C1-C8 amide, C1-C4 sulfonyl, C1-C4 sulfonamide, C1-C4 alkylamino, C1-C5 alkoxy, C3-C6 carbocycle, and 3-10 membered heterocycle,
• wherein the C3-C6 carbocycle is optionally substituted with 1 to 4 substituents selected from hydroxy, halo, and carboxy;
• wherein the 3-10 membered heterocycle is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, —S(O)20H, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycle, and C1-C7 alkyl, wherein the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, C2-C8 ester, and C1-C5 alkoxy;
• Rc is a C3-C6 carbocycle optionally substituted with 1 to 4 substituents selected from hydroxy halo, and carboxy;
• Rd is C1-C4 sulfonyl or C1-C4 sulfonamide;
• Ry is H, C1-C3 alkyl, or C1-3 haloalkyl;
• Rx is H, halo, hydroxy, —CN, —NH2, C1-C3 alkoxy, C1-C3 alkyl, or C1-3 haloalkyl;
• Re is H, halo, C1-C8 alkyl, or C1-C8 haloalkyl;
• X is a C1-C4 alkylene;
• Y is a phenyl or 5-6 membered heteroaryl, wherein the phenyl or heteroaryl is optionally substituted with 1-3 substituents selected from halo, C1-C3 alkyl, C1-C3 alkoxy, —CN, C1-C3 haloalkyl, and cyclopropyl;
• G is N or CH;
• Ga and Gb are N, CH, or CR5 wherein one, and only one, of Ga and Gb is N or CH and one, and only one, of Ga and Gb is CR5;
• R5 is
• Za and Zb are, independently, C1-C3 alkyl or C1-C3 haloalkyl, or Za and Zb form a 3-6 membered carbocycle or heterocycle;
• and
• Zc is H, —CN, C1-C3 alkyl, C1-C3 haloalkyl, or C2-C4 alkyne, or combinations thereof.

A method of inserting a polynucleotide into a genome of a eukaryotic cell, the method comprising:

• a. adding an inhibitor of the microhomology-mediated end joining (MMEJ) pathway to a composition comprising the eukaryotic cell,
• b. transfecting the eukaryotic cell with:
• i. a vector encoding a Cas effector protein,
• ii. a vector comprising a polynucleotide of interest,
• iii. a vector comprising a polynucleotide comprising:
• an RNA guide sequence;
• a Cas-binding region;
• a DNA template sequence, or combinations thereof,
• wherein the vector of (i), (ii) and (iii) can be on the same vector or different vectors, and wherein the polynucleotide of interest is inserted into the genome by homology directed repair (HDR) or single-stranded template repair (SSTR),
• wherein the inhibitor of the MMEJ pathway is an inhibitor of PolQ, selected from a compound of formula (I):
• or any stereoisomer thereof or pharmaceutically acceptable salt thereof;
• wherein,
• R1 and R2 are each, independently, H, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 hydroxyalkyl, —CN, C2-C4 alkyne, or C2-C6 alkoxyalkyl;
• Q1, Q2, and Q3 are, independently N, C-L-R, or CRx, wherein no more than one of Q1, Q2, and Q3 is C-L-R;
• L is a bond, —O—;
• —C(O)—;
• —O(CH2)pC(O)—;
• —C(O)NRy—;
• —O(CH2)pC(O)NRy—;
• —O(CH2)pNRy;
• —NRy—;
• —(CH2)p—;
• —(CH2)pNRy—;
• —(CH2)pO—;
• —(CH2)pC(O)—;
• —(CH2)pC(O)O—;
• —O(CH2)p—;
• p is, independently, 1, 2, or 3
• R is H, Ra, Rb, Rc, or Rd;
• Ra is a 3-10 membered heterocycle optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, —S(O)2OH, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycle, and C1-C7 alkyl, wherein the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, C2-C8 ester, and C1-C5 alkoxy;
• Rb is a C1-C7 alkyl, wherein one or two methylene groups from the C1-C7 alkyl are optionally replaced with NRe or O and one or two single bonds in a C2-C7 alkyl chain are optionally replaced with a double or triple bond(s), wherein the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from:
• halo, oxo, hydroxy, carboxyl, amino, —CN, C2-C4 alkynyl, C2-C6 carbamate, C1-C8 amide, C1-C4 sulfonyl, C1-C4 sulfonamide, C1-C4 alkylamino, C1-C5 alkoxy, C3-C6 carbocycle, and 3-10 membered heterocycle,
• wherein the C3-C6 carbocycle is optionally substituted with 1 to 4 substituents selected from hydroxy, halo, and carboxy;
• wherein the 3-10 membered heterocycle is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, —S(O)20H, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycle, and C1-C7 alkyl, wherein the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, —CN, C2-C8 ester, and C1-C5 alkoxy;
• Rc is a C3-C6 carbocycle optionally substituted with 1 to 4 substituents selected from hydroxy halo, and carboxy;
• Rd is C1-C4 sulfonyl or C1-C4 sulfonamide;
• Ry is H, C1-C3 alkyl, or C1-3 haloalkyl;
• Rx is H, halo, hydroxy, —CN, —NH2, C1-C3 alkoxy, C1-C3 alkyl, or C1-3 haloalkyl;
• Re is H, halo, C1-C8 alkyl, or C1-C8 haloalkyl;
• X is a C1-C4 alkylene;
• Y is a phenyl or 5-6 membered heteroaryl, wherein the phenyl or heteroaryl is optionally substituted with 1-3 substituents selected from halo, C1-C3 alkyl, C1-C3 alkoxy, —CN, C1-C3 haloalkyl, and cyclopropyl;
• G is N or CH;
• Ga and Gb are N, CH, or CR5 wherein one, and only one, of Ga and Gb is N or CH and one, and only one, of Ga and Gb is CR5;
• R5 is
• Za and Zb are, independently, C1-C3 alkyl or C1-C3 haloalkyl, or Za and Zb form a 3-6 membered carbocycle or heterocycle;
• and
• Zc is H, —CN, C1-C3 alkyl, C1-C3 haloalkyl, or C2-C4 alkyne, or combinations thereof.