CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo
Fig 2
Generation of Slx2 knockout mice.
A) Schematic of the genomic target sites in the Slx2 gene. B) Analysis of CRISPR/Cas9 system efficiency in mouse embryonic stem (ES) cells by using T7E1 assay. pX330 ligated with different gRNAs were transfected into V6.5 ES cells. Targeted region was PCR amplified and then digested by T7 Endonuclease I. M, marker. NC, negative control, mouse ES cells transfected with GFP plasmid. C) gRNA-1 and gRNA-3 worked in mouse embryos. Sequencing results of mouse embryos injected with Cas9 mRNA and Slx2 gRNA-1 or gRNA-3 were shown. D) Genotyping results of line 1 and line 11 F1 mice. Cas9-mediated indels lead to frame shift of Slx2. Blue color showed the sequence of gRNAs; protospacer adjacent motifs (PAM) were labeled with purple color; red color showed the modified sequences information after targeting. -, nucleic acid base deletion. E) Western blot analysis of SLX2 expression level in testis from line 1 (#1) and line 11 (#11) mice. WT, wild-type.