Repositorium
Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9
488
Journal Article / 2017
Greiner, Andre; Kelterborn, Simon; Evers, Heide; Kreimer, Georg; Sizova, Irina; Hegemann, Peter
The fast-growing biflagellated single-celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4 (encoding ChR2), COP5, PHOT, UVR8, VGCC, MAT3, and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1, chr2, and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
1195 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
Zinc-finger nucleases ChR1 |
Function as light-gated ion channels SDN3 |
Basic research Basic research |
1196 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
Zinc-finger nucleases ChR2 |
Function as light-gated ion channels SDN3 |
Basic research Basic research |
1197 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 PSY1 |
Albino phenotype SDN2 |
Basic research Basic research |
1198 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 ChR2 |
Function as light-gated ion channels SDN2 |
Basic research Basic research |
1199 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 COP1/2 |
No information SDN2 |
Basic research Basic research |
1200 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 COP5 |
No information SDN2 |
Basic research Basic research |
1201 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 PHOT |
No information SDN2 |
Basic research Basic research |
1202 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 PSY1 |
Albino phenotype SDN2 |
Basic research Basic research |
1203 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 COP1/2-a |
No information SDN3 |
Basic research Basic research |
1204 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 COP5 |
No information SDN3 |
Basic research Basic research |
1205 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 VGCC |
No information SDN3 |
Basic research Basic research |
1206 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 MAT3 |
No information SDN3 |
Basic research Basic research |
1207 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 aCRY-a |
No information SDN3 |
Basic research Basic research |
1208 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 aCRY-b |
No information SDN3 |
Basic research Basic research |
1209 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 UVR8 |
No information SDN3 |
Basic research Basic research |
1210 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 KU80 |
No information SDN3 |
Basic research Basic research |
1211 |
Greiner, Andre; Sizova, Irina Germany |
Chlamydomonas reinhardtii |
CRISPR/Cas9 POLQ |
No information SDN3 |
Basic research Basic research |