Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

Abstract

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.

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

“…While using longer homologous regions in the editing template appeared to improve the yield of edited cells (0.38 Ϯ 0.05 CFU g Ϫ1 DNA for pCas9gRNA-delcac824I-1000 versus 0.2 Ϯ 0.0 CFU g Ϫ1 DNA for pCas9gRNA-delcac824I-500), the transformation efficiency remained low. Toxicity associated with Cas9 has been implicated in several previous reports (53)(54)(55), and this can significantly reduce the transformation efficiency of C. acetobutylicum. Additionally, numerous attempts to construct E. coli-Clostridium vectors with cas9 and sgRNA expression that was regulated by strong, constitutive, and endogenous clostridial promoters, such as thiolase or ferredoxin promoters, yielded only E. coli transformants with deletions/mutations in the resulting plasmids (data not shown).…”

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

“…Furthermore, specific gene modification through zinc-finger nucleases has not proven practical, as it requires time-consuming screens for zinc finger arrays with high specificity for sequences of interest (Sizova et al, 2013). Cas9 coupled with specific guide sequences has been used in multiple systems for high-efficiency genome editing (Cong et al, 2013;Jinek et al, 2013;Mali et al, 2013), but to date has not been successfully used to target gene disruptions in Chlamydomonas (Jiang et al, 2014). While RNA silencing through the use of artificial microRNAs has been developed for Chlamydomonas, suppression of transcript levels is usually incomplete (Molnar et al, 2009;Zhao et al, 2009).…”

Genetic tools and techniques for Chlamydomonas reinhardtii

“…In contrast to positive selectable markers, negative selectable markers represent factors that confer sensitivity towards a certain compound once expressed in the host cell and can be useful e.g., to identify trans-acting factors (Young and Purton 2014) or as markers for gene silencing approaches (Jiang et al 2014;Rohr et al 2004). Three established negative selectable marker for C. reinhardtii are the endogenous MAA7 gene (encoding the tryptophan synthase ß subunit; presence leads to sensitivity to 5-fluoroindole) (Rohr et al 2004), the endogenous FKBP12 gene (encoding the 12-kD FK506-binding protein; presence leads to sensitivity to rapamycin) (Crespo et al 2005), and the E. coli codA gene (encoding cytosine deaminase; presence leads to sensitivity to 5-fluorocytosine) (Young and Purton 2014).…”