|United States Of America||Published Application||20220315914||10/06/2022||2019-162|
Mutated versions of Cas12a that remove its non-specific ssDNA cleavage activity without affecting site-specific double-stranded DNA cutting activity. These mutant proteins, in which a short amino acid sequence is deleted or changed, provide improved genome editing tools that will avoid potential off-target editing due to random ssDNA nicking.
Genome editing in animals, plants, and human cells.
Accurate and efficient genome editing.
Background: Cas12a (formerly called Cpf1) is a type V CRISPR-Cas enzyme derived from bacteria that is used for RNA-guided genome editing in animal, plant and human cells. However, Cas12a possesses an additional enzymatic activity in which a DNA target-bound Cas12a can rapidly and non-specifically degrade any single-stranded DNA (ssDNA) substrate in a sequence-independent manner. This enzymatic activity is endonucleolytic, which means that the ssDNA substrate does not need a free 5' or 3' end to be cut. For this reason, natural Cas12a-type enzymes have the potential to induce off-target genome editing due to nicking of exposed ssDNA in cells.
genome editing, Cas12a