CC-5712 ku80 [line #1]

$30.00

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.