File S1 - Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9

Figure S1. The pUC57-sgRNA expression vector. The sgRNA expression vector was constructed using the backbone of the pUC57 vector with a Kanamycin resistance gene. The annealed oligos were inserted between the two Bsa I restriction sites (blue) downstream of the T7 promoter (red). The construct was linearized by Dra I (green) for in vitro transcription. Figure S2. Cas9:sgRNA-mediated 4 gene modifications by a mixture of 4 single sgRNAs. (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 15 potential founder rats (#1∼15) derived from co-microinjection of a mixture of 4 single sgRNAs as described in Table S2 in File S1. Primers used for PCR amplication were described in Table S3 in File S1. Figure S3. Phenotypes of the mutant potential founder rats. (a) Hematobiochemical assay of wild-type control and potential founder #38 harboring bi-allelic ApoE mutation. The levels of CHO, TG, HDL, and LDL in serum of founder #38 were quantified. The LDL increased up to 275.5% compared with wild-type control rats. CHO, total cholesterol; TG, triglycerides; HDL, high density lipoprotein; LDL, low density lipoprotein. (b) Western blot analysis of B2M expression in potential founder #36 harboring bi-allelic B2m mutations. The expression of B2M in lung of potential founder #36 was not detected by Western blot. (c) Flowcytometry analysis of peripheral blood nucleated cells from wild-type control and founder #31 harboring bi-allelic Prkdc mutation. Dot plots represent CD3, CD45RA positive cells for mature T and B cell subpopulations, respectively. Figure S4. Analysis of the off-target effect. Detection of Cas9:sgRNA-mediated off-target mutation in potential founders #25, #7, #8, #30, #39, and #40 by T7EN1 cleavage assay. Marker and wild-type control were located at the left two lanes of the gel. Samples with different pattern of cleavage bands compared with wild-type control were marked with asterisks and sub-cloned for sequencing. Sequence results showed, except OTS-4 of Prkdc, all the cleavage activities were induced by single nucleotide polymorphism (SNP), genetic variation (Prkdc OTS-37, 27 bp-deletion) or mutations introduced by PCR amplification. Cleavage activities detected in wild-type and potential founders (#) were further confirmed by sequencing. The results showed the mutations were induced by Taq encountering repeat sequence. Figure S5. Analysis of the transmission of the on-target mutation. To analyze the transmission of mutations, potential founders with one (founder #3), two (founder #19), or three (founder #26) mutant genes were selected to cross with wild-type SD rat. (a) Detection of Cas9:sgRNA-mediated on-target cleavage of the endogenous B2m in 8 F1 pups derived from potential founder #3 by T7EN1 cleavage assay. Mutations were detected in 3 F1 pups (1, 4, and 5). (b) DNA sequences of genomic loci in F1 pups 1, 4 and 5. PCR amplicon of the targeted fragment at the B2m in potential founder #3-derived F1 pups 1, 4, and 5 were cloned and sequenced. Sequencing result showed one kind of mutation same as the founder #3 was detected in the offspring, indicating that mutations induced by Cas9:sgRNA was transmittable. However, 351 bp-deletion mutants can't be detected in offspring, suggesting that Cas9 function may not only in one-cell, but also in the later stage. (c) Detection of Cas9:sgRNA-mediated on-target cleavage of the endogenous B2m and Prkdc in 12 F1 pups derived from potential founder #19 by T7EN1 cleavage assay. The mutations were detected in all 12 F1 pups. (d) DNA sequences of genomic loci in mutant pups. PCR amplicon of the targeted fragment at the B2m and Prkdc in potential founder #19-derived F1 pups were cloned and sequenced. Sequencing result showed the mutations same as the founder #19 were detected in the offspring. Two kinds of mutation of Prkdc were all transmittable, indicating mosaicism induced by Cas9:sgRNA. (e) Detection of Cas9:sgRNA-mediated on-target cleavage of the endogenous ApoE by PCR, B2m and Prkdc by T7EN1 cleavage assay in 10 F1 pups derived from potential founder #26. The mutations were detected in F1 pups. (f) DNA sequences of genomic loci in mutant pups. Smaller band of PCR amplicon of ApoE were gel extracted and sequenced. PCR amplicon of the targeted fragment at the B2m and Prkdc in potential founder #26-derived F1 pups were cloned and sequenced. Sequencing result showed the mutations same as the founder #26 were detected in the offspring. Table S1. Oligonucleotides for generating sgRNA expression vectors. Table S2. Summary of embryo injections of sgRNA:Cas9. Table S3. Primers for amplifying sgRNA targeted loci. Table S4. Summary of mutations of multiple genes. Table S5. Summary of the alleles for putative off-target sites. Table S6. Primers for amplifying off-target sites.
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