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Cpf1 represents a novel single RNA‐guided CRISPR/Cas endonuclease system suitable for genome editing with distinct features compared with Cas9. We demonstrate the functionality of three Cpf1 orthologues –  Acidaminococcus spp.  BV3L6 (AsCpf1),  Lachnospiraceae bacterium  ND2006 (LbCpf1) and  Francisella novicida  U112 (FnCpf1) – for genome editing of   Saccharomyces cerevisiae  . These Cpf1‐based systems enable fast and reliable introduction of donor DNA on the genome using a two‐plasmid‐based editing approach together with linear donor DNA. LbCpf1 and FnCpf1 displayed editing efficiencies comparable with the CRISPR/Cas9 system, whereas AsCpf1 editing efficiency was lower. Further characterization showed that AsCpf1 and LbCpf1 displayed a preference for their cognate crRNA, while FnCpf1‐mediated editing with similar efficiencies was observed using non‐cognate crRNAs of AsCpf1 and LbCpf1. In addition, multiplex genome editing using a single LbCpf1 crRNA array is shown to be functional in yeast. This work demonstrates that Cpf1 broadens the genome editing toolbox available for   Saccharomyces cerevisiae  . © 2017 The Authors.  Yeast  published by John Wiley & Sons, Ltd.

CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae