Generation of multiplex genetic modified rats using CRISPR/Cas9 system.

<p>(a) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> by T7EN1 cleavage assay. PCR amplicon of the targeted fragment at the <i>ApoE</i>, <i>B2m</i>, <i>Prf1</i>, and <i>Prkdc</i> in 15 founder rats (#1∼15) were subjected to T7EN1 cleavage assay. Founder #13, which is quadruple gene mutant, was marked with asterisks. (b) DNA sequences of four loci in founder #13. PCR amplicon with cleaved bands in T7EN1 cleavage assay were cloned and sequenced. The PAM sequence was underlined and highlighted in green; the targeting site are red; the mutations are blue, lower case; insertions (+) or deletions (−) are shown to the right of each allele.