CRISPR/Cas9 mutagenesis in Volvox carteri.

Volvox carteri and other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis in V.?carteri using CRISPR/Cas9 components expressed from transgenes. We used V.?carteri nitrate reductase gene (nitA) regulatory sequences to conditionally express Streptococcus pyogenes Cas9， and V.?carteri U6 RNA gene regulatory sequences to constitutively express single-guide RNA (sgRNA) transcripts. Volvox carteri was bombarded with both Cas9 vector and one of several sgRNA vectors programmed to target different test genes (glsA， regA and invA)， and transformants were selected for expression of a hygromycin-resistance marker present on the sgRNA vector. Hygromycin-resistant transformants grown with nitrate as sole nitrogen source (inducing for nitA) were tested for Cas9 and sgRNA expression， and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sgRNA plasmid with glsA protospacer sequence yielded progeny (at a rate of ~0.01%) with a gonidialess (Gls) phenotype similar to that observed for previously described glsA mutants， and sequencing of the glsA gene in independent mutants revealed short deletions within the targeted region of glsA， indicative of Cas9-directed non-homologous end joining. Similarly， bombardment of a morphologically wild-type strain with the Cas9 plasmid and sgRNA plasmids targeting regA or invA yielded regA and invA mutant transformants/progeny， respectively (at rates of 0.1-100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development in V.?carteri， and of developmental novelty in the volvocine algae.