Plasmids

From Don Weeks, University of Nebraska, April 2017

pWEN7 is ampicillian resistant in E. coli and has the complete DNA sequence of the Cas9/intron-sgRNA targeting the FKB12 gene in Chalmy.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)

From Don Weeks, University of Nebraska, April 2017

pWEN8 is ampicillian resistant in E. coli and has the complete DNA sequence of the Cas9/intron-sgRNA targeting the ARG gene in Chlamy.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)

From Luke Mackinder/Martin Jonikas, University of York, UK/Princeton, April 2017

This plasmid was designed for generating C-terminal CrYFP-3xFLAG fusion proteins. The plasmid can be cut with HpaI and then gene of interest inserted by Gibson assembly.

Selection: ampicillin in E. coli, paromomycin in Chlamydomonas

host strain: DH5α

Sequence details: https://www.ncbi.nlm.nih.gov/nuccore/KX077945.1

Sequence, FASTA & ApE files

Yang W, Wittkopp TM, Li X, Warakanont J, Dubini A, Catalanotti C, Kim RG, Nowack ECM, Mackinder LCM, Aksoy M, Dudley Page M, D’Adamo S, Saroussi S, Heinnickel M, Johnson X, Richaud P, Alric J, Boehm M, Jonikas MC, Benning C, Merchant S, Posewitz M, Grossman AR (2015) Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism. Proc Natl Acad Sci 112:14978-14983

Mackinder LCM, Meyer MT, Mettler-Altmann T, Chen V, Mitchell MC, Caspari O, Freeman Rosenzweig ES, Pallesen L, Reeves G, Itakura A, Roth R, Sommer F, Geimer S, Mühlhaus T, Schroda M, Goodenough U, Stitt M, Griffiths H, Jonikas MC (2016) A repeat protein links Rubisco to form the eukaryotic carbon concentrating organelle. Proc Natl Acad Sci 113:5958-5963

From Luke Mackinder/Martin Jonikas, University of York, UK/Princeton, April 2017

This plasmid was designed for generating C-terminal mCherry-3xHis fusion proteins. The plasmid can be cut with HpaI and then gene of interest inserted by Gibson assembly.

Selection: ampicillin in E. coli, hygromycin in Chlamydomonas

host strain: DH5α

Sequence details: https://www.ncbi.nlm.nih.gov/nuccore/KX077949.1

Sequence, FASTA & ApE files

Mackinder LCM, Meyer MT, Mettler-Altmann T, Chen V, Mitchell MC, Caspari O, Freeman Rosenzweig ES, Pallesen L, Reeves G, Itakura A, Roth R, Sommer F, Geimer S, Mühlhaus T, Schroda M, Goodenough U, Stitt M, Griffiths H, Jonikas MC (2016) A repeat protein links Rubisco to form the eukaryotic carbon concentrating organelle. Proc Natl Acad Sci 113:5958-5963

From James Umen, Donald Danforth Plant Science Center, October 2017

host strain: PIR1
kanamycin resistant

Sequence

López-Paz C, Liu D, Geng S, Umen JG. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression. Plant J. 2017 Oct 4. doi: 10.1111/tpj.13731. [Epub ahead of print] PubMed PMID: 28980350

From James Umen, Donald Danforth Plant Science Center, October 2017

host strain: PIR1
kanamycin resistant

Sequence

López-Paz C, Liu D, Geng S, Umen JG. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression. Plant J. 2017 Oct 4. doi: 10.1111/tpj.13731. [Epub ahead of print] PubMed PMID: 28980350

From James Umen, Donald Danforth Plant Science Center, October 2017

host strain: PIR1
kanamycin resistant

Sequence

López-Paz C, Liu D, Geng S, Umen JG. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression. Plant J. 2017 Oct 4. doi: 10.1111/tpj.13731. [Epub ahead of print] PubMed PMID: 28980350

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: CrVENUS, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: sfGFP-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: CrmCherry, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: CrmCherry-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: PSAD (with RBCS2 1st intron)
Insert: CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: TUB2 (with RBCS2 1st intron)
Insert: CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2 (with RBCS2 1st intron, ATG mutated to CTG)
Insert: CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

unpublished

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: PSAD (with RBCS2 1st intron, ATG mutated to CTG)
Insert: CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

unpublished

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: TUB2 (with RBCS2 1st intron, ATG mutated to CTG)
Insert: CrVENUS-3FLAG, APHVII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR (2016) Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda) 6:4115-4125

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: Lifeact-CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR, Cross FR (2015) Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas. Genetics 202:977-96

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: NAP1gDNA, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR, Cross FR (2015) Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas. Genetics 202:977-96

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: TUB2 (with RBCS2 1st intron)
Insert: NAP1gDNA, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Onishi M, Pringle JR, Cross FR (2015) Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas. Genetics 202:977-96

From Masayuki Onishi, John Pringle lab, Stanford University School of Medicine, October 2017

Promoter: HSP70A/RBCS2
Insert: DUS16-3HA-2gp64-3FLAG-CrVENUS-3FLAG, APHVIII

host strain: Mach1
ampicillin resistant

SnapGene file

Yamasaki T, Onishi M, Kim EJ, Cerutti H, Ohama T (2016) RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 113:10720-5

From Simon Kelterborn, Peter Hegemann lab, Humboldt University-Berlin October 2017

Expression of Streptococcus pyogenes (Sp)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A heat-shock promoter.

Host strain: XL1-Blue
Selection in E.coli: ampicillin
Link to sequence and map
Sequence (.docx file)

Overview of all CRISPR/Cas9 plasmids from the Hegemann lab

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de

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. Plant Cell 30 (2017). https://doi.org/10.1105/tpc.17.00659

From Simon Kelterborn, Peter Hegemann lab, Humboldt University-Berlin October 2017

Expression of Streptococcus pyogenes (Sp)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A/RBCS2 tandem promoter.

Host strain: XL1-Blue
Selection in E. coli: ampicillin
Link to sequence and map
Sequence (.docx file)

Overview of all CRISPR/Cas9 plasmids from the Hegemann lab

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de

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. Plant Cell 30 (2017). https://doi.org/10.1105/tpc.17.00659

From Simon Kelterborn, Peter Hegemann lab, Humboldt University-Berlin October 2017

Expression of Staphylococcus aureus (Sa)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A heat-shock promoter.

Host strain: XL1-Blue
Selection in E. coli: ampicillin
Link to sequence and map
Sequence (.docx file)

Overview of all CRISPR/Cas9 plasmids from the Hegemann lab

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de

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. Plant Cell 30 (2017). https://doi.org/10.1105/tpc.17.00659

From Simon Kelterborn, Peter Hegemann lab, Humboldt University-Berlin October 2017

Expression of Staphylococcus aureus (Sa)Cas9 optimized to C. reinhardtii codon bias, controlled by the HSP70A/RBCS2 tandem promoter.

Host strain: XL1-Blue
Selection in E. coli: ampicillin
Link to sequence and map
Sequence (.docx file)

Overview of all CRISPR/Cas9 plasmids from the Hegemann lab

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de

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. Plant Cell 30 (2017). https://doi.org/10.1105/tpc.17.00659

From Simon Kelterborn, Peter Hegemann lab, Humboldt University-Berlin October 2017

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6snRNA promoter #1.

Host strain: DH10b
Selection in E. coli: ampicillin
Link to sequence and map
Sequence (.docx file)

Overview of all CRISPR/Cas9 plasmids from the Hegemann lab

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de

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. Plant Cell 30 (2017). https://doi.org/10.1105/tpc.17.00659

From Simon Kelterborn, Peter Hegemann lab, Humboldt University-Berlin October 2017

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #2.

Host strain: DH10b
Selection in E. coli: ampicillin
Link to sequence and map
Sequence (.docx file)

Overview of all CRISPR/Cas9 plasmids from the Hegemann lab

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de

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. Plant Cell 30 (2017). https://doi.org/10.1105/tpc.17.00659

From Simon Kelterborn, Peter Hegemann lab, Humboldt University-Berlin October 2017

Cloning vector for Streptococcus pyogenes Cas9 (SpCas9) single guide RNA transcription, controlled by the U6 snRNA promoter #3 (3’ of Cre08.g371751). The 20 bp target site can be inserted in a cut-ligation reaction as annealed oligos into an Esp3I cloning site following the protocol of Ann Ran (Ran et al. 2013). The immediate 4-bp sequence upstream of the transcriptional start site was changed to ACTT in all U6 constructs to simplify the cloning procedures.

Host strain: DH10b
Selection in E. coli: ampicillin
Link to sequence and map
Sequence (.docx file)

Overview of all CRISPR/Cas9 plasmids from the Hegemann lab

Visit www.chlamy.de for more info or contact CRISPR@chlamy.de

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. Plant Cell 30 (2017). https://doi.org/10.1105/tpc.17.00659

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 8:2281-2308