Generation of Helq Knockout Mice

Helq^−/− mice were generated by CRISPR/Cas9 technology. Briefly, one single-guide RNA (sgRNA) were designed targeting the first exon of Helq and cloned into pSpCas9(BB)-2A-GFP (PX458) vector as previously described^{107 (link)}. The designed plasmid was electroporated into mouse BVSC ESCs using Neon^TM Transfection System (Thermofisher) according to the manufacturer’s instructions. Two days later, GFP-positive cells were sorted by FACS and transferred manually into Matrigel-coated 96-well-plates with one single cell per well. Genomic DNA was extracted from the harvested cells and used for Sanger Sequencing. The targeted ESCs with 8-nt deletion in the two alleles of Helq were injected into blastocysts to generate chimeric mice. The chimeric mice were self-crossed to generate Helq^−/− mice. Genomic DNA was extracted to confirm the genotype.