Abstract
CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA–target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position for PAM TATV and at position for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.
Highlights
CRISPR/Cas systems are adaptive immune systems found in bacteria and archaea that defend prokaryotes against bacteriophages and other mobile genetic elements deemed to be revolutionary tools for genetic engineering
To characterize the editing activities of multiple Cas12a orthologues and variants, we adopted and modified the high-throughput evaluation methods applied to Cas9 and
To provide more available options for Cas12a effectors in addition to the most employed As- and LbCas12a, here, we expanded the Cas12a toolkit by introducing three new Cas12a variants engineered from potential candidates in the high-throughput activity profiling of 16 Cas12a orthologues
Summary
CRISPR/Cas systems are adaptive immune systems found in bacteria and archaea that defend prokaryotes against bacteriophages and other mobile genetic elements deemed to be revolutionary tools for genetic engineering. Compared with Cas, endonucleases from the Cas12a family exhibit various distinct features: (i) more compact structures; (ii) guidance by a single short crRNA without the need to transactivate CRISPR RNA; (iii) both DNase and RNase activities, and CRISPR array processing; (iv) recognition of a target with T-rich protospacer adjacent motifs (PAMs) located at the 5 end; (v) generation of PAM-distal staggered overhangs; and (vi) relatively low off-target effects [5,6] These properties make Cas12a effectors promising when targeting AT-rich genomic regions applied for multiplexed gene editing and applications requiring high specificity [7,8,9,10,11]. Screening for potential Cas12a candidates and engineering them for improved editing efficiency and broader PAM compatibility are needed
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