The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 
system has become a powerful and precise tool for targeted gene modification 
(e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. 
Initial attempts to apply this technology to a model, the single-cell alga, 
Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To 
determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. 
reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one 
of four different sgRNAs to cause targeted gene disruption during a 24-h period 
immediately following transformation. All three exogenously supplied gene 
targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. 
reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as 
determined by DNA sequencing of cloned PCR amplicons of the target site region. 
Success in transient expression of Cas9 and sgRNA genes contrasted with the 
recovery of only a single rapamycin-resistant colony bearing an appropriately 
modified FKB12 target site in 16 independent transformation experiments 
involving >10(9) cells. Failure to recover transformants with intact or 
expressed Cas9 genes following transformation with the Cas9 gene alone (or even 
with a gene encoding a Cas9 lacking nuclease activity) provided strong 
suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. 
reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA 
genes function properly in C. reinhardtii to cause targeted gene modifications 
and point to the need for a focus on development of methods to properly stem 
Cas9 production and/or activity following gene editing.