Figure 4.

Complicated gene manipulation techniques. A, Point mutation: Co‐injecting a single‐stranded DNA (ssDNA) or double‐stranded DNA (dsDNA) can cause a single nucleotide base insertion, deletion, or substitution through homologous recombination (HR). B, Tag insertion: tags, such as a FLAG‐tag, can be inserted similarly to point mutations by using ssDNA or dsDNA and HR. Reporter genes, such as enhanced green fluorescent protein (EGFP), can also be knocked in. C, Double knockout: by using a combination of different sgRNAs, Cas9 can cleave multiple target genes to generate complex KO mice. D, Large deletion: the CRISPR/Cas9 system can mediate large genomic deletions by transfecting two or more sgRNAs