Significance
          
            Our findings establish a method of efficient, targeted genome editing in
            Chlamydomonas reinhardtii
            . We demonstrate an approach to bypass inefficient gene targeting via homologous recombination and achieve homology-directed DNA replacement in
            C. reinhardtii
            . In addition, we report CRISPR/Cpf1-mediated DNA editing efficiencies being boosted 500-fold through the use of single-stranded oligodeoxynucleotides (ssODNs) as repair templates. It remains to be determined whether Cpf1-induced staggered DNA cleavage enhances ssODN-mediated gene editing in a wider range of species and whether the underlying repair pathway(s) responsible is more broadly conserved.
          </jats:p

Ferenczi, Aron

Molnar, Attila

Pyott, Douglas Euan

Xipnitou, Andromachi

Proceedings of the National Academy of Sciences

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 ∼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

eScholarship - University of California

English

Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA

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 ∼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.</p

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