CRISPR /Cas9 mutagenesis in Volvox carteri

Summary 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 (sg RNA ) transcripts. Volvox carteri was bombarded with both Cas9 vector and one of several sg RNA 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 sg RNA vector. Hygromycin‐resistant transformants grown with nitrate as sole nitrogen source (inducing for nitA ) were tested for Cas9 and sg RNA expression, and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sg RNA 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 sg RNA 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.