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

Authors: Swiat, Michal A.; Dashko, Sofia; den Ridder, Maxime; Wijsman, Melanie; van der Oost, John; Daran, Jean Marc; Daran-Lapujade, Pascale;

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.

Country
Netherlands
Keywords

Gene Editing, 570, Endodeoxyribonucleases, Saccharomyces cerevisiae, Life Science, Point Mutation, CRISPR-Cas Systems, Francisella, Genome, Fungal, Synthetic Biology and Bioengineering, Repetitive Sequences, Nucleic Acid

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
views
OpenAIRE UsageCountsViews provided by UsageCounts
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129
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9
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gold