FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae. Issue 21 (2nd November 2017)

• Record Type: Journal Article
• Authors: Świat, Michal A.; Dashko, Sofia; den Ridder, Maxime; Wijsman, Melanie; van der Oost, John; Daran, Jean-Marc; Daran-Lapujade, Pascale
• Is Part Of: Nucleic acids research. Volume 45:Issue 21(2017)
• Page Start: 12585
• Page End: 12598
• Publication Date: 2017-11-02
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkx1007
• Languages: English
• ISSNs: 0305-1048

Abstract

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 Fn Cpf1 can mediate RNA-guided DNA cleavage at targeted genomic loci in the popular model and industrial yeast Saccharomyces cerevisiae. Fn Cpf1 very efficiently and precisely promoted repair DNA recombination with efficiencies up to 100%. Furthermore, Fn Cpf1 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 Fn Cpf1 was able to edit four genes simultaneously in yeast with a 100% efficiency. A remarkable observation was the unexpected, strong preference of Fn Cpf1 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 Fn Cpf1 expression and easy construction of crRNA expression cassettes in S. cerevisiae. Fn Cpf1 proves to be a powerful addition to S. cerevisiae CRISPR toolbox.