Systems, methods, and compositions for site-specific genetic engineering using programmable addition via site-specific targeting elements (paste)
AssigneeMassachusetts Institute of Technology
InventorsOmar Abudayyeh, Jonathan Gootenberg
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Abstract
This disclosure provides systems, methods, and compositions for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE). PASTE comprises the addition of an integration site into a target genome followed by the insertion of one or more genes of interest or one or more nucleic acid sequences of interest at the site. PASTE combines gene editing technologies and integrase technologies to achieve unidirectional incorporation of genes in a genome for the treatment of diseases and diagnosis of disease.
Claims25
A system capable of site-specifically integrating an exogenous nucleic acid into a mammalian cell genome at a desired target site, wherein the system comprises, in a single composition:
- (a) a nucleic acid encoding a DNA binding nickase domain linked to a reverse transcriptase domain;
- (b) a nucleic acid encoding at least two guide RNAs (gRNAs) comprising, from 3′ to 5′,
- i. a primer binding sequence,
- ii. a sequence complementary to one strand of an integration recognition sequence, and
- iii. a target binding sequence,
- wherein the gRNA is capable of guiding the linked nickase-reverse transcriptase domains to the genomic target site;
- (c) nucleic acid encoding an integration enzyme;
- and
- (d) an exogenous nucleic acid linked to a sequence that is an integration cognate of the integration recognition sequence.
Dependencies are inferred from the claim text.