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Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM). However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT ( T ethering EX onuclease T 5 with FnCas12a)-a fusion strategy that significantly increased the knockout efficiency of FnCas12a in human cells at multiple genomic loci in three different cell lines. TEXT results in higher insertion and deletion efficiency than FnCas12a under different spacer lengths from 18 nt to 23 nt. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. Compared to other Cas12a orthologs, including AsCas12a and LbCas12a, TEXT achieves the highest on-targeting efficiency and shows minimal off-targeting effects at all tested sites. TEXT enhances the activity of FnCas12a nuclease and expands its targeting scope and efficiency in human cell genome engineering.

Improving FnCas12a Genome Editing by Exonuclease Fusion