FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae

Abstract

Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can mediate RNA-guided DNA cleavage at targeted genomic loci in the popular model and industrial yeast Saccharomyces cerevisiae. FnCpf1 very efficiently and precisely promoted repair DNA recombination with efficiencies up to 100%. Furthermore, FnCpf1 was shown to introduce point mutations with high fidelity. While editing multiple loci with Cas9 is hampered by the need for multiple or complex expression constructs, processing itself a customized CRISPR array FnCpf1 was able to edit four genes simultaneously in yeast with a 100% efficiency. A remarkable observation was the unexpected, strong preference of FnCpf1 to cleave DNA at target sites harbouring 5′-TTTV-3′ PAM sequences, a motif reported to be favoured by Cpf1 homologs of Acidaminococcus and Lachnospiraceae. The present study supplies several experimentally tested guidelines for crRNA design, as well as plasmids for FnCpf1 expression and easy construction of crRNA expression cassettes in S. cerevisiae. FnCpf1 proves to be a powerful addition to S. cerevisiae CRISPR toolbox.

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

“…Instead of G-rich PAMs required by Cas9, Cas12a recognizes T-rich PAMs and thus further increases the number of potential target sites. Recent publications in yeast and rice have revealed that a PAM of 5 0 -TTTV-3 0 is also strongly preferred by FnCas12a [69,70]. In contrast to Cas9 which generates bluntended DSBs proximal to the PAM, Cas12a generates staggered DSBs distal from the PAM.…”

“…The Cas12a orthologues recognize T-rich PAMs, which are 5 0 -TTV(A/C/ G)-3 0 for FnCas12a and 5 0 -TTTV-3 0 for AsCas12a and LbCas12a, although 5 0 -TTN-3 0 and 5 0 -TTTN-3 0 can also be recognized at lower efficiency [68]. Recent publications in yeast and rice have revealed that a PAM of 5 0 -TTTV-3 0 is also strongly preferred by FnCas12a [69,70]. Recently, the targeting range for Cas12a has been expanded greatly by engineered versions that recognize 5 0 -TYCV-3 0 and 5 0 -TATV-3 0 PAMs without sacrificing efficiency or specificity [71].…”

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

“…However, the current CRISPR/ Cas9-based multiplex genome editing tool for P. patens typically requires simultaneous delivery of multiple gRNA plasmids (Lopez-Obando et al, 2016;Nomura et al, 2016), making it complex and time-consuming for plasmid construction. Although Cas12a-mediated gene editing has been intensively studied in several plant species such as rice (Kim et al, 2016;Zetsche et al, 2016;Hu et al, 2017;Swiat et al, 2017;Tang et al, 2017;Wang et al, 2017;Ding et al, 2018;Li et al, 2018b), this technique has not previously been employed in moss. Since the efficiency of gene targeting by Cas12a varies from species to species (Tang et al, 2017;Lee et al, 2018), the CRISPR/Cas12a system requires further optimization.…”

Multiplex gene editing in rice with simplified CRISPR‐Cpf1 and CRISPR‐Cas9 systems

“…However, a later study demonstrated that to achieve high cleavage activity in human cells, FnCpf1 requires a PAM defined as 5’‐YTV‐3’ (Y represents C or T, and V represents A, C or G), and the spacer sequence with a length of 21 nt, rather than the commonly used 23‐25 nt, could lead to maximal editing (Tu et al ). Another study further showed that FnCpf1 preferred the TTTV motif to cleave target DNA when expressed in Saccharomyces cerevisiae (Świat et al ). Based on these reports, we modified some of the guides and cloned the array into the TCTU expression cassette, together with hammerhead and HDV ribozyme (Figure A; Table S2).…”