Repositorium
Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii
89
Journal Article / 2014
Jiang, Wenzhi; Brueggeman, Andrew J.; Horken, Kempton M.; Plucinak, Thomas M.; Weeks, Donald P.
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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
178 |
Weeks, Donald P. USA |
Chlamydomonas reinhardtii |
CRISPR/Cas9 FKB12 |
hygromycin resistance gene SDN1 |
Basic research Basic research |
179 |
Weeks, Donald P. USA |
Chlamydomonas reinhardtii |
CRISPR/Cas9 Gluc |
mutant luciferase SDN1 |
Basic research Basic research |
180 |
Weeks, Donald P. USA |
Chlamydomonas reinhardtii |
CRISPR/Cas9 Hygro |
hygromycin resistance gene SDN1 |
Basic research Basic research |
181 |
Weeks, Donald P. USA |
Chlamydomonas reinhardtii |
CRISPR/Cas9 mGFP |
mutant green fluorescence protein SDN1 |
Basic research Basic research |