Figure 1.
Generation of multiplex genetic modified rats using CRISPR/Cas9 system.
(a) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous ApoE, B2m, Prf1, and Prkdc by T7EN1 cleavage assay. PCR amplicon of the targeted fragment at the ApoE, B2m, Prf1, and Prkdc 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.
Figure 2.
Schematic diagrams of sgRNAs and DNA sequences of targeting genomic loci.
PCR amplicon of the targeted fragment in the ApoE, B2m, Prf1, and Prkdc in all founder rats (#1∼40) were sequenced. The PAM sequence is underlined and highlighted in green; the targeting site are red; the mutations are blue, lower case; insertions (+), deletions (−) or mutant (m) are shown to the right of each allele. N/N indicates positive colonies out of total sequenced. (a) ApoE locus. (b) B2m locus. (c) Prf1 locus. (d) Prkdc locus.
Table 1.
Summary of the mutation of the founder rats.
Figure 3.
Cas9:sgRNA-mediated modifications in 4 genes by a mixture of dual sgRNAs for each gene.
(a) PCR identification of sgRNA:Cas9-mediated site-specific cleavage of the endogenous ApoE, B2m, Prf1, and Prkdc loci. The genetic modification analysis was performed by PCR amplification of the targeted fragment in the ApoE, B2m, Prf1, and Prkdc in 25 founder rats (#16∼40) derived from co-microinjection of a mixture of dual sgRNAs for each genes as described in Table S2 in File S1. Primer used for PCR amplication was described in Table S3 in File S1. Additionally, founder #36, #38 had a larger deletion in the ApoE, the primer ApoE-NS2 and ApoE-NAS2 used for amplification. Founder #38 had a larger deletion in the B2m, the primer B2m-S2 and B2m-AS2 used for amplification. (b) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous ApoE, B2m, Prf1, and Prkdc by T7EN1 cleavage assay. PCR products from (a) were subjected to T7EN1 cleavage assay as described in material and methods.
Figure 4.
Analysis of the transmission of the off-target mutation.
(a) Detection of Cas9:sgRNA-mediated off-target cleavage of Prkdc OTS-4 in all founders (#1–40) by T7EN1 cleavage assay. PCR amplicon of Prkdc OTS-4 in all 40 founder rats were subjected to T7EN1 cleavage assay as described in methods. Total 23 founders (*) displayed cleavage bands. (b) PCR products with cleavage bands were cloned and sequenced. Sequence result showed OTS-4 indeed mutagenized in the 23 founders. Indels were also detected around 270 bp downstream of the OTS-4 in most colonies, which may be introduced by PCR amplification when Taq encountering repeat sequence. (c) Detection of Cas9:sgRNA-mediated off-target cleavage of Prkdc OTS-4 in 8 F1 pups derived from founder #3 by T7EN1 cleavage assay. Mutations were detected in 2 F1 pups (4 and 8). (d) DNA sequences of Prkdc OTS-4 in F1 pups 4 and 8. PCR amplicon of the Prkdc OTS-4 in founder #3-derived F1 pups 4 and 8 were sequenced. Sequencing result showed one kind of off-target mutation same as the founder #3 was detected in the offspring, indicating that off-target mutation induced by Cas9:sgRNA was heritable.