Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing

Abstract

Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.

Figure 1

Validation of each sgRNA sequence using marmoset fibroblast cells. (A) Schema of linearized target genes including exons (black boxes) and single-guide RNA (sgRNA) positions (white arrows). The marmoset c-kit gene (top) is located on chromosome 3 and contains 21 exons. 579 glutamic acid (E579, GAG) is located on exon 11 (yellow box). Marmoset Shank3 is located on chromosome 1 and contains 22 exons and 1227 alanine (A1227, GCC) is located on exon 21 (yellow box). (B) Cleavage activities of each sgRNA in marmoset fibroblast cells. The target gene modification by each sgRNA with hCas9 was examined using marmoset fibroblast cells and CEL-1 assay to confirm cleavage activity of designed sgRNAs. Green arrowheads indicate shifted bands compared to negative control, indicating target gene modification by CEL-1 assay. M; size marker, NC; negative control – PCR product obtained using wild-type marmoset tissue as a PCR template. (C) Rate of modified sequence clones in each target gene by subcloning and sequence analysis using PCR products of marmoset fibroblast cells.

Figure 2

Validation of CRISPR/Cas9 cleavage activity in marmoset embryo. (A) Flowchart of target gene modification validation using CRISPR/Cas9 injected whole embryos. The embryos were cultured in vitro, and collected into PCR tubes directly after zona pellucida removal. Subsequently, PCR was performed on the collected embryos. (B) A portion of the CEL-1 assay result using PCR product obtained from CRISPR/Cas9 injected embryos. Upper panels show the results of c-kit targeted CRISPR/mRNA (mRNA) injected embryos (a), and CRISPR/nuclease (nuclease) injected embryos (b). Lower panels show the results of Shank3 targeted CRISPR/Cas9 injected embryos (c,d). Lanes 1–5 in all panels contain Cel-1 nuclease digested DNA of PCR products obtained from each CRISPR/Cas9 injected embryo. M; size marker, NC; negative control – PCR product obtained using wild-type marmoset tissue as a PCR template. (C) Representative Sanger sequencing chromatograms of sub-clones derived from CRISPR/Cas9-injected embryos. Left panels show the results of sub-clones obtained from c-kit-targeted CRISPR/mRNA (mRNA Embryo3) or CRISPR/nuclease (nuclease Embryo3)-injected embryos. Right panels show the results of sub-clones obtained from Shank3-targeted CRISPR/Cas9-injected embryos (mRNA Embryo3 and nuclease Embryo3). This figure correlates with embryo samples listed in Supplemental Table 7 (c-kit) and 8 (Shank3). The top of each panel exhibits wild-type sequences of each target gene. Gray arrow; sgRNA sequence, red arrow; the position of indel or substitution mutations in each target gene.

Figure 3

Blastomere analysis. (A) Flowchart used for each blastomere obtained from CRISPR/Cas9 injected embryos. The embryos were cultured, the zona pellucida was removed, and blastomeres were split. Each blastomere was collected into a PCR tube, and used for PCR analysis. (B) The CEL-1 assay of blastomeres from the embryo containing the c-kit gene modified by CRISPR/nuclease injection (Embryo2, Supplementary Table 11) in upper panels. Left panel shows the results of the CEL-1 assay of each blastomere, and right panel shows the result using a mixture of PCR products obtained from blastomere and wild-type tissues to detect homozygotic modification. Arrowheads indicate heterozygotic modification (green) and homozygotic modification (red) in the target gene. Lane 1–8 in both panels contain Cel-1 nuclease digested DNA obtained from PCR products of each blastomere. M; size marker, NC; negative control – PCR product obtained using wild-type marmoset tissue as a PCR template. Lower panels show the Sanger sequencing chromatograms (left of lower panels) and the sequences (right of lower panels) of these blastomeres. Both top lines indicate wild-type sequences of target genes. Red or green arrowheads of the left end of the lower panel are correlated with the results of the CEL-1 assay in the upper panels. Black box; inserted sequence into c-kit, black arrow; position of the 1-bp deletion modification, gray arrow; sgRNA sequence. (C) Quantitative results of target gene modification obtained from blastomere analyses. The graph shows the percentage of blastomeres containing intact (blue), bi-allelic modifications (red) and mono-allelic modifications (green) for each injection condition and target gene.

Figure 4

c-kit gene targeted Knock-in analysis by single blastomeres. (A) The ratio of each modified blastomere of 36 nt ssODN and CRISPR/nuclease injection KI embryos is shown. Circles represent an injected embryo in each condition, and each color indicates the blastomere sequence. Red indicates a precise KI homozygous blastomere, blue indicates heterozygous blastomere of precise KI and wild type, green represents a heterozygous precise KI and KO (indel) mutation blastomere, and yellow indicates homozygous or heterozygous KO blastomeres. Embryo number (no) s in this illustration are correlated to the Embryo no. in Supplementary Tables 14 and 15. KI: knock-in, KO: knock-out. (B) Representative Sanger sequencing chromatograms of modified c-kit and putative amino acid sequences derived from blastomere sequence analyses of c-kit in the KI experiment correlate with the blastomere samples listed in Supplemental Table 15. (a) Wild-type blastomere (Embryo 1, 36nt-AS and CRISPR/nuclease injection). (b) KI blastomere (Embryo 2, 36nt-S and CRISPR/nuclease injection). Blue and red arrows indicate knock-in of donor ssODN, red arrows and letters indicate mutations contributing to E579K mutant (W³⁷). (c) KI/wild-type heterozygous blastomere (Embryo 3, 36nt-S and CRISPR/nuclease injection). (d) Imprecise KI blastomere (Embryo 1, 36nt-S and CRISPR/nuclease injection). The top sequences indicate the KI donor ssODN. The insertion (black arrows) caused a frameshift mutation and introduced a stop codon (*).