Saccharomyces cerevisiae IMX1139 (Fncpf1)

Cpf1, a class II CRISPR-Cas endonuclease like Cas9, is a new family of 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 Cpf1 can mediate RNA-guided DNA cleavage at targeted endogenous genomic loci. Cpf1 can very efficiently and precisely promote donor DNA recombination with efficiencies up to 100%. Using homologous recombination repair, Cpf1 can also be used to introduce point mutations with high fidelity. While targeting of multiple genomic loci with Cas9 is hampered by the need for multiple or complex expression constructs, using the ability of Cpf1 to process its own CRISPR RNA (crRNA) two genes were edited simultaneously in yeast with a hundred percent efficiency. A surprizing observation is the inability of FnCpf1 to cleave DNA at target sites harbouring 5’VTTV3’and 5’-TTTN-3’ PAM. When expressed in S. cerevisiae, FnCpf1 favoured the 5’-TTTV-3’ PAM reported for its relatives from 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 is a promising addition to S. cerevisiae CRISPR toolbox.