Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 114, Issue 51, Pages 13567-13572Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1710597114
Keywords
Chlamydomonas reinhardtii; CRISPR/Cpf1; RNP; ssODN; editing
Categories
Funding
- Biotechnology and Biological Sciences Research Council (BBSRC) PHYCONET Proof of Concept Fund [PHYCPoC-31]
- BBSRC East of Scotland BioScience (EASTBIO) National Productivity Investment Fund (NPIF) Industrial Cooperative Awards in Science & Technology (CASE) studentship [BB/R505493/1]
- BBSRC EASTBIO [BB/J01446X/1]
- Biotechnology and Biological Sciences Research Council [1311408] Funding Source: researchfish
- BBSRC [1941078] Funding Source: UKRI
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The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as similar to 10% efficiency in C. reinhardtii. We demonstrate its use in transgene-and selection-free generation of sequence-specific mutations and epitope tagging at an endogenous locus. As the direct delivery of gene-editing reagents bypasses the use of transgenes, this method is potentially applicable to a wider range of species without the need to develop methods for stable transformation.
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