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US 12,767,959 B2Granted
Nucleobase editors having reduced off-target deamination and methods of using same to modify a nucleobase target sequence
AssigneeBeam Therapeutics Inc.
InventorsNicole Gaudelli, Yi Yu, Ian Slaymaker, Jason Michael Gehrke, Seung-Joo Lee, David A. Born
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Abstract
The invention features nucleobase editors and multi-effector nucleobase editors having an improved editing profile with minimal off-target deamination, compositions comprising such editors, and methods of using the same to generate modifications in target nucleobase sequences.
Claims27
A cytidine base editor fusion protein comprising:
- (i) a polynucleotide programmable DNA binding domain, and
- (ii) a cytidine deaminase comprising SEQ ID NO:
- 5 having an alteration selected from the group consisting of R33A, K34A, R52A, and H122A, further comprising between zero (0) and twenty (20) amino acid alterations at positions selected from the group consisting of positions 15 to 198, and comprising a F at amino acid position 120,
- wherein the cytidine base editor fusion protein has an increased ratio of in cis to in trans activity (in cis:in trans) as compared to a standard cytidine base editor fusion protein comprising a rat APOBEC-1 cytidine deaminase (rAPOBEC-1) and the polynucleotide programmable DNA binding domain,
- wherein the cytidine deaminase does not comprise SEQ ID NO:
- 4, and
- wherein “in cis” activity refers to on-target DNA base editing wherein the cytidine base editor is directed by a guide RNA, and “in trans” activity refers to unguided off-target editing events wherein the cytidine base editor is not directed by the guide RNA.
4 more elements
A cytidine base editor fusion protein comprising:
- (i) a polynucleotide programmable DNA binding domain, and
- (ii) a cytidine deaminase comprising SEQ ID NO:
- 5 with an alteration selected from the group consisting of R33A, K34A, R52A, and H122A, further comprising between zero (0) and twenty (20) amino acid alterations at positions selected from the group consisting of positions 15, 16, 21, 28, 30, 36, 52, 53, 60, 62, 88, 90, 118, 121, 126, 128, 130, 132, 169, and 198,
- wherein the cytidine base editor fusion protein has an increased ratio of in cis to in trans activity (in cis:in trans) as compared to a standard cytidine base editor fusion protein comprising a rat APOBEC-1 cytidine deaminase (rAPOBEC-1) and the polynucleotide programmable DNA binding domain,
- wherein the cytidine deaminase does not comprise SEQ ID NO:
- 4, and
- wherein “in cis” activity refers to on-target DNA base editing wherein the cytidine base editor is directed by a guide RNA, and “in trans” activity refers to unguided off-target editing events wherein the cytidine base editor is not directed by the guide RNA.
4 more elements
A cytidine base editor fusion protein comprising:
- (i) a polynucleotide programmable DNA binding domain, and
- (ii) a cytidine deaminase comprising SEQ ID NO:
- 5 with an alteration selected from the group consisting of R33A, K34A, R52A, and H122A,
- wherein the cytidine base editor fusion protein has an increased ratio of in cis to in trans activity (in cis:in trans) as compared to a standard cytidine base editor fusion protein comprising a rat APOBEC-1 cytidine deaminase (rAPOBEC-1) and the polynucleotide programmable DNA binding domain,
- wherein the cytidine deaminase does not comprise SEQ ID NO:
- 4, and
- wherein “in cis” activity refers to on-target DNA base editing wherein the cytidine base editor is directed by a guide RNA, and “in trans” activity refers to unguided off-target editing events wherein the cytidine base editor is not directed by the guide RNA.
4 more elements
Dependencies are inferred from the claim text.