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. 2018 Oct 24;98(1):36–45. doi: 10.1177/0022034518805978

Figure 5.

Figure 5.

Applications of CRISPR/Cas9 to generate mutant animals. (A) General scheme to generate mutant mouse models. DNAs or RNAs encoding Cas9 and single-guide RNA (sgRNA) with or without template DNA are microinjected into the pronuclei of the fertilized egg. These modified zygotes are reimplanted to the oviduct of pseudo-pregnant surrogate mice. After 19 gestational days, mutant mice offspring are born. (B) Generation of floxed animal models. The left arm represents the simultaneous method. This method involves coinjecting nucleotides encoding Cas9, 2 sgRNAs, and 2 individual loxP template DNAs to zygote. It simultaneously knocks in 2 loxP alleles to flank the target exon gene. However, large deletions can occur as a result. The right arm represents the sequential method. This method involves coinjecting Cas9, first sgRNA, and first loxP template DNA at 1–cell stage embryo to knock in the first loxP allele. On the next day, the second group of Cas9, second sgRNA, and second loxP template DNA are injected into 2–cell stage embryos, leading to knock-in of the second loxP allele.