Homology-Directed Transgene-Free Gene Editing in Chlamydomonas reinhardtii

Abstract

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 realized. 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 20% as a proportion of total cells (Ferenczi et al. Proc Natl Acad Sci U S A 114:13567-13572, 2017). 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.