The green alga 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 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 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.

Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.

Chlamydomonas reinhardtii is a microalgal model organism with a suite of molecular and genetic techniques, but routine editing of its nuclear genome is yet to be realised. DNA-based transformation techniques are prohibitively inefficient and lead to predominantly nonhomologous (i.e. off-target) integration. Standard CRISPR-based gene editing protocols have proved too ineffective to enable routine application. We have found that the use of CRISPR/Cpf1 in conjunction with single-stranded DNA (ssODN) repair templates achieves nuclear gene editing efficiencies as high as 30% [1]. This produces edits with predictable outcomes in a transgene-and selection marker-free manner. The possibility to purchase all necessary reagents commercially with no preparation time (besides design) facilitates rapid and routine genetic engineering in this organism. Here we describe the use of this technique to knockout locus FKB12, which leads to rapamycin resistance and lends itself to an easy assay when adopting this gene-editing protocol.