CC-5717 polq1 [line #3]

$30.00

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.