Plasmids deposited by the Hegemann lab at the Chlamydomonas Resource Center
Visit www.chlamy.de for more info or contact CRISPR@chlamy.de. Please verify your plasmids by sequencing.
Streptomyces aminoglycoside-5 ́- phosphotransferase VIII (aphVIII) antibiotic selection marker gene for paromomycin selection, optimized to C. reinhardtii codon bias. map (GenBank; updated 09/2019)
Streptomyces aminoglycoside phosphotransferase (aphVII) marker gene for Hygromycin B selection, optimized to C. reinhardtii codon bias. map (GenBank)
For recombinant Cas9 protein expression in E. coli, we use plasmid pET-28b-Cas9-His (addgene #47327) of the Alex Schier Lab and their protocol for purification.
Expression of Streptococcus pyogenes (Sp)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A heat-shock promoter. map
Expression of Streptococcus pyogenes (Sp)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A/RBCS2 tandem promoter. map
Expression of Staphylococcus aureus (Sa)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A heat-shock promoter. map
Expression of Staphylococcus aureus (Sa)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A/RBCS2 tandem promoter. map
pCrU6.1-SpCas9 cloning (pPH195)
Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #1. map
pCrU6.2-SpCas9 cloning (pPH158)
Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #2. map
pCrU6.3-SpCas9 cloning (pPH159)
Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #3. map
pCrU6.4-SpCas9 cloning (pPH160)
Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #4. map
pCrU6.4-SpCas9 cloning/aphVIII (pPH340)
Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map
pCrU6.4-SpPSY1/aphVIII (pPH332)
Vector for single guide RNA targeting PSY1 with RNA scaffold for Streptococcus pyogenes Cas9 (SpCas9), controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map
pCrU6.1-SaCas9 cloning (pPH193)
Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #1. map
pCrU6.2-SaCas9 cloning (pPH154)
Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #2. map
pCrU6.3-SaCas9 cloning (pPH155)
Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #3. map
pCrU6.4-SaCas9 cloning (pPH156)
Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #4. map
pCrU6.4-SaCas9 cloning/aphVIII (pPH339)
Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map
pCrU6.4-SaPSY1/aphVIII (pPH331)
Vector for guide RNA targeting PSY1 with scaffold for Staphylococcus aureus Cas9 (SaCas9), controlled by the U6 snRNA promoter #4 with aphVIII cassette for selection on paromomycin. map
Reference
Greiner A, Kelterborn S, Evers H, Kreimer G, Sizova I, and Hegemann P. Targeting of photoreceptor genes via zinc-finger nucleases and CRISPR/Cas9 in Chlamydomonas reinhardtii. The Plant Cell October 2017. https://doi.org/10.1105/tpc.17.00659