Dh5α competent e coli strain

The procedures of CRISPR-Cas9 gene editing to knockout mdig were as reported previously [22 (link)]. Briefly, to generate the CRISPR-Cas9 plasmid, mdig CDS sequence was supplied into the CRISPR Design tool (http://crispr.mit.edu/, accessed on 17 July 2022), and single guide RNA (sgRNA) sequence targeting exon 3 of mdig was selected. The sense and antisense primer sequences are 5′-CACCGAATGTGTACATAACTCCCGC-3′ and 5′-AAACGCGGGAGTTATGTACACATTC-3′, respectively. Single-stranded sense and antisense primers were annealed to form double-strand oligos at 95 °C for 5 min, and then cooled down to 25 °C for 5 min. Vector pSpCas9-2A-Blast was digested with BpiI (BbsI) restriction enzymes (Thermo Fisher Scientific, Ann Arbor, MI, USA). sgRNA pairs and linearized vector were ligated by T4 DNA ligase (Thermo Fisher Scientific, Ann Arbor, MI, USA) for 10 min at 22 °C. Then the ligation product was transferred into DH5α competent E. coli strain (Thermo Fisher Scientific, Ann Arbor, MI, USA) according to the manufacturer’s protocol.

To generate the CRISPR-Cas9 plasmid, mdig CDS sequence was inputted into the CRISPR Design tool (http://crispr.mit.edu/), and single guide RNA (sgRNA) sequence targeting on exon 3 of mdig was selected. The sense and antisense primer sequences are 5'-CACCGAATGTGTACATAACTCCCGC-3' and 5'-AAACGCGGGAGTTATGTACACATTC-3', respectively. Single-stranded sense and antisense primers were annealed to form double-strand oligos in 95 °C for 5 min, and then cooled down to room temperature at a speed of 5°C/min. Vector pSpCas9-2A-Blast was digested with BpiI (BbsI) restriction enzymes (Thermo Fisher Scientific, Ann Arbor, MI). sgRNA pairs and linearized vector were ligated by T4 DNA ligase (Thermo Fisher Scientific) for 10 min at 22°C. Then the ligation product was transferred into DH5α competent E.coli strain (Thermo fisher scientific) according to the manufacturer's protocol.