Fig. 1

XIST gene related information and gene knockout strategy A. XIST gene size and the size and location of each chromosome.B. A-F on vertical coordinate represents 7 repeat sequences of the XIST gene, the locations of repeated sequences in mice, voles, rats, moles, cattle, dogs and humans are showed in the figure, and the location of exon 1.C. Three knockout schemes of XIST gene were designed: One was the knockout of the first exon; The second is the knockout of all exons in segments; The third option is the knockout of the entire XIST gene"

Table 1

sgRNA designed for XIST gene target sites"

Fig. 2

Efficiency identification and cutting efficiency of sgRNA after electrotransfection A. sgRNA efficiency after electrotransfection, dark blue represents GFP negative cells, pink represents GFP positive cells. The conversion efficiency is 43.3%. B. The sgRNA sites of the 3 knockout models, red is the PAM region, scheme one is to knockout the first exon; The second is to knockout all exons in segments; The third option is to knockout the entire XIST gene. C. The successful knockout zone and knockout efficiency statistics of each sgRNA. D. The 5 off-target sites predicted by sgRNA, namely OT1, OT2, OT3, OT4 and OT5, the knockout effect of off-target sites was verified. E. The mismatch site was verified by T7E1, and no cutting site was found"

Table 2

sgRNA efficiency statistics"

Fig. 3

Identification of electrotransfection efficiency and monoclonal cell line culture A. Expression of GFP fluorescent protein 24 h after electrotransfection, green is GFP fluorescence expression (100X). B. The efficiency of sgRNA after electrotransfection was 39.9%, with dark blue representing GFP negative cells and pink representing GFP positive cells. C. Cell status in 48-and 24-well plates of cultured monoclones (100X)"

Table 3

Monoclonal culture and identification  板·个-1"

Fig. 4

Genotype identification and knockout efficiency of monoclonal cell lines A. M. DNA molecular weight standard; 250-500 bp white bands are homozygous knockout cell lines, marked by red arrows. The 2 000 bp white bands represent cell lines that were not successfully knocked out. B. M. DNA molecular weight standard; The first 5 DNA bands represent homozygous knockout cell lines, and the last DNA band represents heterozygous knockout cell lines. C. Sequencing of homozygous mutant cell lines, red is the knockout region, the results are consistent with PCR identification"