Abstract
ABSTRACT CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5’-TTTV-3’ protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a’s gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5’-TTTV-3’ PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5’-NYHV-3’ PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.
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ABSTRACT
CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5’-TTTV-3’ protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a’s gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5’-TTTV-3’ PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5’-NYHV-3’ PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.
Competing Interest Statement
The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. J.A.D. is a cofounder of Azalea Therapeutics, Caribou Biosciences, Editas Medicine, Evercrisp, Scribe Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member at Evercrisp, Caribou Biosciences, Scribe Therapeutics, The Column Group and Inari. She also is an advisor for Aditum Bio. J.A.D. is Chief Science Advisor to Sixth Street, a Director at Johnson & Johnson, Altos Labs and Tempus AI, and has a research project sponsored by Apple Tree Partners.
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