Strains

From Francis Fields, Stephen Mayfield lab, University of California-San Diego, June 2021

Wild isolate from the UCSD Biology Field Station from a pond of HSM media. Grows on TAP or HSM. Mating type unknown. Tried many times to generate gametes but was unsuccessful. No bands when probed with C. reinhardtii mating type primers. This was originally named 002wt when sent to the Chlamy Center.

Fields FJ, Hernandez RE, Weilbacher E, Garcia-Vargas E, Huynh J, Thurmond M, Lund R, Burkart MD, Mayfield SP (2021) Annual productivity and lipid composition of native microalgae (Chlorophyta) at a pilot production facility in Southern California. Algal Research, 56:102307. https://doi.org/10.1016/j.algal.2021.102307

From Francis Fields, Stephen Mayfield lab, University of California-San Diego, June 2021

Wild isolate from the UCSD Biology Field Station from a pond of HSM media. Grows on TAP or HSM. Mating type confirmed via mating with CC-124. This was originally named 010wt when sent to the Chlamy Center.

Fields FJ, Hernandez RE, Weilbacher E, Garcia-Vargas E, Huynh J, Thurmond M, Lund R, Burkart MD, Mayfield SP (2021) Annual productivity and lipid composition of native microalgae (Chlorophyta) at a pilot production facility in Southern California. Algal Research, 56:102307. https://doi.org/10.1016/j.algal.2021.102307

From Francis Fields, Stephen Mayfield lab, University of California-San Diego, June 2021

Wild type CC library pooled and mated with each other. Resulting progeny selected for tolerance to high salt (>300 mM salt). This strain was isolated from the surviving variants and shows grows well in the presence of salt, but cells tend to clump. Grows on TAP or HSM. Grows on nitrate. Mating type confirmed via mating with CC-124.

From Francis Fields, Stephen Mayfield lab, University of California-San Diego, June 2021

Wild isolate of Chlamydomonas found actively mating in wet soil at the UCSD Biology Field Station. This isolate is the mating partner to 403wt, strains appear to be isogamous and heterothallic like C. reinhardtii and mate reliably when following standard mating protocols. Strain has demonstrated tolerance to high pH (>10), grows better than C. reinhardtii in outdoor conditions, and is readily transformable. Grows on TAP or HSM. Grows on nitrate.

Opposing mating partner to CC-5699. No bands when probed with C. reinhardtii mating type primers. Initial tests suggest infertile with C. reinhardtii but not conclusive (only tested against CC-124/125).

From Francis Fields, Stephen Mayfield lab, University of California-San Diego, June 2021

See CC-5697 [402wt] for background information. Strain was transformed via electroporation and expresses mClover fluorescent protein in the nucleus. Grows on TAP or HSM. Grows on nitrate.

From Francis Fields, Stephen Mayfield lab, University of California-San Diego, June 2021

Wild isolate of Chlamydomonas found actively mating in wet soil at the UCSD Biology Field Station. This isolate is the mating partner to 402wt, strains appear to be isogamous and heterothallic like C. reinhardtii and mate reliably when following standard mating protocols. Strain has demonstrated tolerance to high pH (>10), grows better than C. reinhardtii in outdoor conditions, and is readily transformable. Grows on TAP or HSM. Grows on nitrate.

Opposing mating partner to CC-5697. No bands when probed with C. reinhardtii mating type primers. Initial tests suggest infertile with C. reinhardtii but not conclusive (only tested against CC-124/125).

From Francis Fields, Stephen Mayfield lab, University of California-San Diego, June 2021

CC library pooled and subjected to UV mutagenesis. Resulting mutants were plated on TAP and grown in the dark. A red colored colony was observed and isolated. Cells appear colorless with red granules. The insoluble red substance was observed accumulating outside the cells as well.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of brca2 (Cre13.g566900) in CC-1883, resulting in deficiency of homologous recombination (HR) DNA repair. One of three brca2 lines deposited (CC-5701, CC-5702, CC-5703).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

HR deficiency was confirmed by hypersensitivity to DNA-damaging agents zeocin and mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 8). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of brca2 (Cre13.g566900) in CC-1883, resulting in deficiency of homologous recombination (HR) DNA repair. One of three brca2 lines deposited (CC-5701, CC-5702, CC-5703).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

HR deficiency was confirmed by hypersensitivity to DNA-damaging agents zeocin and mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 8). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of brca2 (Cre13.g566900) in CC-1883, resulting in deficiency of homologous recombination (HR) DNA repair. One of three brca2 lines deposited (CC-5701, CC-5702, CC-5703).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

HR deficiency was confirmed by hypersensitivity to DNA-damaging agents zeocin and mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 8). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of rad51a (Cre14.g622850) in CC-1883, resulting in deficiency of homologous recombination (HR) DNA repair. One of three rad51a lines deposited (CC-5704, CC-5705, CC-5706).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

HR deficiency was confirmed by hypersensitivity to DNA-damaging agents zeocin and mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 8). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of rad51a (Cre14.g622850) in CC-1883, resulting in deficiency of homologous recombination (HR) DNA repair. One of three rad51a lines deposited (CC-5704, CC-5705, CC-5706).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

HR deficiency was confirmed by hypersensitivity to DNA-damaging agents zeocin and mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 8). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of rad51a (Cre14.g622850) in CC-1883, resulting in deficiency of homologous recombination (HR) DNA repair. One of three rad51a lines deposited (CC-5704, CC-5705, CC-5706).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

HR deficiency was confirmed by hypersensitivity to DNA-damaging agents zeocin and mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 8). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of fancd2 (Cre10.g453650) in CC-1883, resulting in deficiency of Fanconi anaemia (FA) DNA repair. One of three fancd2 lines deposited (CC-5707, CC-5708, CC-5709).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

FA deficiency was confirmed by hypersensitivity to the DNA-damaging agent mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 9). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of fancd2 (Cre10.g453650) in CC-1883, resulting in deficiency of Fanconi anaemia (FA) DNA repair. One of three fancd2 lines deposited (CC-5707, CC-5708, CC-5709).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

FA deficiency was confirmed by hypersensitivity to the DNA-damaging agent mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 9). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of fancd2 (Cre10.g453650) in CC-1883, resulting in deficiency of Fanconi anaemia (FA) DNA repair. One of three fancd2 lines deposited (CC-5707, CC-5708, CC-5709).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

FA deficiency was confirmed by hypersensitivity to the DNA-damaging agent mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 9). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of fancm (Cre03.g208833) in CC-1883, resulting in deficiency of Fanconi anaemia (FA) DNA repair.

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

FA deficiency was confirmed by hypersensitivity to the DNA-damaging agent mitomycin C, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 9). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of ku70 (Cre13.g607500) in CC-1883, resulting in deficiency of non-homologous end-joining (NHEJ) DNA repair. Three ku80 lines are also deposited (CC-5712, CC-5713, CC-5714).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

NHEJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 10). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Note: since KU70 and KU80 are part of the dimeric KU70/80 complex, we have used ku70 and ku80 mutants collectively as ku70/80 mutants to investigate NHEJ-deficiency (Ferenczi et al. 2021).

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of ku80 (Cre10.g423800) in CC-1883, resulting in deficiency of non-homologous end-joining (NHEJ) DNA repair. One of three ku80 lines deposited (CC-5712, CC-5713, CC-5714). One ku70 line is also deposited.

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

NHEJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig.10). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Note: since KU70 and KU80 are part of the dimeric KU70/80 complex, we have used ku70 and ku80 mutants collectively as ku70/80 mutants to investigate NHEJ-deficiency (Ferenczi et al. 2021).

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of ku80 (Cre10.g423800) in CC-1883, resulting in deficiency of non-homologous end-joining (NHEJ) DNA repair. One of three ku80 lines deposited (CC-5712, CC-5713, CC-5714). One ku70 line is also deposited.

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

NHEJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig.10). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Note: since KU70 and KU80 are part of the dimeric KU70/80 complex, we have used ku70 and ku80 mutants collectively as ku70/80 mutants to investigate NHEJ-deficiency (Ferenczi et al. 2021).

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of ku80 (Cre10.g423800) in CC-1883, resulting in deficiency of non-homologous end-joining (NHEJ) DNA repair. One of three ku80 lines deposited (CC-5712, CC-5713, CC-5714). One ku70 line is also deposited.

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

NHEJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig.10). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Note: since KU70 and KU80 are part of the dimeric KU70/80 complex, we have used ku70 and ku80 mutants collectively as ku70/80 mutants to investigate NHEJ-deficiency (Ferenczi et al. 2021).

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of polq1 (Cre16.g664301) in CC-1883, resulting in deficiency of alternative end-joining (alt-EJ) DNA repair. One of three polq1 lines deposited (CC-5715, CC-5716, CC-5717).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

Alt-EJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 11). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of polq1 (Cre16.g664301) in CC-1883, resulting in deficiency of alternative end-joining (alt-EJ) DNA repair. One of three polq1 lines deposited (CC-5715, CC-5716, CC-5717).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

Alt-EJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 11). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of polq1 (Cre16.g664301) in CC-1883, resulting in deficiency of alternative end-joining (alt-EJ) DNA repair. One of three polq1 lines deposited (CC-5715, CC-5716, CC-5717).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

Alt-EJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 11). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Note: this line has a 9 bp deletion at the polq1 locus (see Ferenczi et al. 2021, Supplementary Fig. 11), so it harbours at least one off-target aphVIII insertion elsewhere in the genome since it is paromomycin resistant.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of polq2 (Cre08.g384390) in CC-1883, resulting in deficiency of alternative end-joining (alt-EJ) DNA repair. One of four polq2 lines deposited (CC-5718, CC-5719, CC-5720, CC-5721).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

Alt-EJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 11). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of polq2 (Cre08.g384390) in CC-1883, resulting in deficiency of alternative end-joining (alt-EJ) DNA repair. One of four polq2 lines deposited (CC-5718, CC-5719, CC-5720, CC-5721).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

Alt-EJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 11). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of polq2 (Cre08.g384390) in CC-1883, resulting in deficiency of alternative end-joining (alt-EJ) DNA repair. One of four polq2 lines deposited (CC-5718, CC-5719, CC-5720, CC-5721).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

Alt-EJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 11). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Chew Yen Peng, Aron Ferenczi and Attila Molnar, University of Edinburgh-UK, December 2021

Knock-out of polq2 (Cre08.g384390) in CC-1883, resulting in deficiency of alternative end-joining (alt-EJ) DNA repair. One of four polq2 lines deposited (CC-5718, CC-5719, CC-5720, CC-5721).

Knock-out generated using CRISPR/Cas12a and the 1.8 kb aphVIII antibiotic selection marker to disrupt the gene of interest, resulting in paromomycin resistance (paroR).

Alt-EJ deficiency was confirmed by hypersensitivity to the DNA-damaging agent zeocin, and the knock-out locus was sequence-verified (see Ferenczi et al. 2021, Supplementary Fig. 11). No off-target analysis was performed; may contain off-target edits and aphVIII integrations.

For the Cas12a gRNA target sequence and PCR conditions (incl. primers), see Ferenczi et al. 2021, Supplementary Data 7 and 9, respectively. Up to 10% DMSO may be added to the PCR to help amplify long GC-rich amplicons, in which case lower annealing temperatures by 3–5°C.

Deposited as part of 21 lines containing eight different DNA repair mutant genotypes (CC-5701 to CC-5721). For full list, see Ferenczi et al. 2021, Supplementary Data 10.

Ferenczi A, Chew YP, Kroll E, von Koppenfels C, Hudson A, Molnar A. Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii. Nat Commun. 2021 Nov 19;12(1):6751. doi: 10.1038/s41467-021-27004-1. PMID: 34799578; PMCID: PMC8604939.

From Jun Zhang, William Snell, University of Maryland, College Park, August 2021

HA tagged HAP2 with point mutation W173A/F177A transformed into 40d4 strain.

Feng J, Dong X, Pinello J, Zhang J, Lu C, Iacob RE, Engen JR, Snell WJ, Springer TA. Fusion surface structure, function, and dynamics of gamete fusogen HAP2. Elife. 2018 Oct 3;7:e39772. doi: 10.7554/eLife.39772. PMID: 30281023; PMCID: PMC6170185.

• Locus:
• HAP2
• Chromosome:
• 16