Improving FnCas12a genome editing by exonuclease fusion

Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM), which will help target previously inaccessible genomic sites. 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 (Tethering EXonuclease T5 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 shows higher insertions and deletions (indels) efficiency than FnCas12a using different spacer lengths from 18nt to 23nt, in which 18nt results in highest fold increase, with up to 11 folds higher efficiency than FnCas12a. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. TEXT enhances the activity of FnCas12a nuclease and expand its application in human cell genome engineering.