Efficient targeted DNA editing and replacement inChlamydomonas reinhardtiiusing Cpf1 ribonucleoproteins and single-stranded DNA

Abstract

<p id="d10063776e169">Our findings establish a method of efficient, targeted genome editing in <i>Chlamydomonas reinhardtii</i>. We demonstrate an approach to bypass inefficient gene targeting via homologous recombination and achieve homology-directed DNA replacement in <i>C. reinhardtii</i>. 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. </p><p class="first" id="d10063776e178">The green alga <i>Chlamydomonas reinhardtii</i> 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 <i>Chlamydomonas</i> 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 <i>C. reinhardtii</i>. 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>