Print Email Facebook Twitter FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae Title FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae Author Swiat, M.A. (TU Delft BT/Industriele Microbiologie) Dashko, S. (TU Delft BT/Industriele Microbiologie) den Ridder, M.J. (Student TU Delft) Wijsman, M. (TU Delft BT/Industriele Microbiologie) van der Oost, John (Wageningen University & Research) Daran, J.G. (TU Delft BT/Industriele Microbiologie) Daran-Lapujade, P.A.S. (TU Delft BT/Industriele Microbiologie) Date 2017-12-01 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. To reference this document use: http://resolver.tudelft.nl/uuid:d9673b57-4af8-4a0a-a6da-cf1262777d4b DOI https://doi.org/10.1093/nar/gkx1007 ISSN 0305-1048 Source Nucleic acids research, 45 (21), 12585-12598 Part of collection Institutional Repository Document type journal article Rights © 2017 M.A. Swiat, S. Dashko, M.J. den Ridder, M. Wijsman, John van der Oost, J.G. Daran, P.A.S. Daran-Lapujade Files PDF gkx1007.pdf 1.73 MB Close viewer /islandora/object/uuid:d9673b57-4af8-4a0a-a6da-cf1262777d4b/datastream/OBJ/view