Abstract
Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1’s PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.
Highlights
Some bacterial and archaeal clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases (Cas) have been repurposed for genome modifications [1,2,3,4], transcription modulation [5,6,7,8] and chromosome imaging [9,10,11,12] in eukaryotic cells
To test the cleavage efficiency of Cpf1 nucleases in mammalian cells, we employed a GFxFP reporter assay previously reported (Supplementary Figure S2 and Supplementary Table S2) [19] that is based on the recovery of an interrupted GFP sequence containing about 500 nucleotidelong homologous stretches
The cleavage efficiency of the Lb, As, Mb- and FnCpf1 nucleases was comparable when assessed on the same targets with three-thymidinenucleotide protospacer adjacent motif (PAM) sequence (Figure 1, Supplementary Figure S3); LbCpf1 seems to perform slightly better
Summary
Some bacterial and archaeal clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases (Cas) have been repurposed for genome modifications [1,2,3,4], transcription modulation [5,6,7,8] and chromosome imaging [9,10,11,12] in eukaryotic cells. The area of their application is continuously widening owing to the simplicity of reprogramming their target specificity. They offer several features distinct from Cas nucleases, such as (i) producing 5 overhangs, (ii) utilization of a shorter guide RNA,
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