US 11,834,658 B2
Systems, methods, and compositions for site-specific genetic engineering using programmable addition via site-specific targeting elements (paste)
Omar Abudayyeh, Cambridge, MA (US); and Jonathan Gootenberg, Cambridge, MA (US)
Assigned to Massachusetts Institute of Technology, Cambridge, MA (US)
Filed by Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Dec. 14, 2022, as Appl. No. 18/066,233.
Application 18/066,233 is a continuation of application No. 17/649,308, filed on Jan. 28, 2022, granted, now 11,572,556.
Application 17/649,308 is a continuation of application No. 17/451,734, filed on Oct. 21, 2021, abandoned.
Claims priority of provisional application 63/222,550, filed on Jul. 16, 2021.
Claims priority of provisional application 63/094,803, filed on Oct. 21, 2020.
Prior Publication US 2023/0135673 A1, May 4, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/85 (2006.01); C12N 15/11 (2006.01); A61K 31/7105 (2006.01); C12N 9/12 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/113 (2010.01); C12N 15/90 (2006.01)
CPC C12N 15/111 (2013.01) [A61K 31/7105 (2013.01); C12N 9/1276 (2013.01); C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/113 (2013.01); C12N 15/85 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05); C12N 2310/3519 (2013.01); C12Y 207/07049 (2013.01)] 22 Claims
 
1. A method of site-specifically integrating an exogenous nucleic acid sequence into a mammalian cell genome or intracellular target nucleic acid, the method comprising:
(a) incorporating at least one integration sequence at a specific target site in the cell genome or intracellular target nucleic acid by introducing ex vivo into a mammalian cell:
(i) an expressible polynucleotide construct encoding an editing polypeptide, wherein the editing polypeptide comprises a DNA binding nuclease domain linked via a linker to a reverse transcriptase domain, wherein the DNA binding nuclease domain comprises a nickase activity; and
(ii) at least two guide RNAs (gRNAs), each comprising a targeting sequence, a primer binding sequence, and a complement of the at least one integration sequence,
wherein each of the at least two gRNAs interacts with the expressed editing polypeptide to direct the editing polypeptide to the specific target site of the cell genome or intracellular target nucleic acid,
wherein the DNA binding nuclease domain nicks a strand of the cell genome or intracellular target nucleic acid to form a nicked site, and
wherein the reverse transcriptase domain reverse transcribes the complement of the at least one integration sequence within each of the gRNAs and thereby incorporates the at least one integration sequence into the nicked site, thereby incorporating the at least one integration sequence at the specific target site of the cell genome or intracellular target nucleic acid; and
(b) integrating an exogenous nucleic acid sequence into the cell genome or intracellular target nucleic acid by introducing into the cell:
(i) the exogenous nucleic acid sequence linked to a sequence that is an integration cognate to the at least one site-specifically incorporated-integration sequence; and
(ii) an expressible polynucleotide construct encoding an integration enzyme, wherein the integration enzyme integrates the exogenous nucleic acid sequence into the cell genome or the intracellular target nucleic acid at the at least one site-specifically incorporated integration sequence, thereby site-specifically integrating the exogenous nucleic acid sequence into the cell genome or the intracellular target nucleic acid,
wherein the expressible polynucleotide encoding the editing polypeptide, the at least two gRNAs, the expressible polynucleotide construct encoding the integration enzyme, and the exogenous nucleic acid sequence are introduced into the mammalian cell concurrently.