Efficient targeted DNA editing and replacement in <i>Chlamydomonas reinhardtii</i> using Cpf1 ribonucleoproteins and single-stranded DNA.

Abstract

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.