Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases
1
has been hindered by the requirement for
an extended TTTV protospacer adjacent motif (PAM)
2
. To address this limitation, we
engineered an enhanced
Acidaminococcus sp.
Cas12a variant
(enAsCas12a) that has a substantially expanded targeting range, enabling
targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits
two-fold higher genome editing activity on sites with canonical TTTV PAMs
compared to wild-type AsCas12a, and we successfully grafted a subset of
mutations from enAsCas12a onto other previously described AsCas12a
variants
3
to enhance
their activities. enAsCas12a improves the efficiency of multiplex gene editing,
endogenous gene activation, and C-to-T base editing, and we engineered a
high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target
effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary
human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively,
enAsCas12a provides an optimized version of Cas12a that should enable wider
application of Cas12a enzymes for gene and epigenetic editing. [AU: Revised
abstract OK?]
Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
Prime Editors have been delivered using DNA or mRNA vectors. Here we demonstrate prime editing (PE) with purified ribonucleoprotein (RNP) complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and pegRNA scaffold incorporations. In HEK293T and primary human T cells, PE RNP complexes introduced desired edits with frequencies of up to 21% and 7.5%, respectively.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.