Efficient targeted DNA editing and replacement in <i>Chlamydomonas reinhardtii</i> 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.