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Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Wang X, Zhou J, Cao C, Huang J, Hai T, Wang Y, Zheng Q, Zhang H, Qin G, Miao X, Wang H, Cao S, Zhou Q, Zhao J - Sci Rep (2015)

Bottom Line: Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks.The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%.Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

No MeSH data available.


R1 sgRNA-induced site-specific insertion with ssODN through HDR.A.(A) schematic of the targeting site with the ssODN sequence used to introduce an exogenous KpnI sequence (underlined). The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from individual blastocyst digested with KpnI restriction enzymes. Three out of 23 blastocysts demonstrated site-specific KpnI sequence insertion through HDR. (C) Sequence analysis of the three blastocysts at the target site. Two blastocysts showed precise HDR-based addition of the KpnI sequence. One blastocyst showed precise addition at the 3′ end, and 117 bp indels were noted at the 5′ side of the modification site. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. KpnI sequence is underlined.
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f3: R1 sgRNA-induced site-specific insertion with ssODN through HDR.A.(A) schematic of the targeting site with the ssODN sequence used to introduce an exogenous KpnI sequence (underlined). The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from individual blastocyst digested with KpnI restriction enzymes. Three out of 23 blastocysts demonstrated site-specific KpnI sequence insertion through HDR. (C) Sequence analysis of the three blastocysts at the target site. Two blastocysts showed precise HDR-based addition of the KpnI sequence. One blastocyst showed precise addition at the 3′ end, and 117 bp indels were noted at the 5′ side of the modification site. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. KpnI sequence is underlined.

Mentions: The DSBs mediated by CRISPR/Cas9 can stimulate a homologous recombination in the presence of a DNA donor with the appropriate homology arms. Recent works have demonstrated that single-stranded DNA oligonucleotides (ssODNs) can be used as substitutes for conventional plasmid-based targeting vectors as donor templates for homology-directed repair (HDR)36. Because of its high mutagenesis efficiency, we hypothesized that R1 sgRNA could assist in HDR. Hence, we co-injected Cas9 mRNA, sgRNA and ssODN containing 6 bp KpnI restriction site flanked by 26 bps homologous sequences on each side into mature MII pig oocytes (Fig. 3A). After parthenogenetic activation, oocytes were cultured to the blastocyst stage and single blastocysts were picked for genotyping. In these experiments, the RFLP assays, as shown in Fig. 3B, identified 3 out of 23 R1 sgRNA-injected blastocysts carrying the KpnI site at the target locus, indicating R1 sgRNA yielded an HDR efficiency as high as 13.04% with ssODN at a concentration of 80 ng/μL. Subsequent sequence analyses indicated two precise KpnI site insertions, which demonstrated successful targeted restriction site insertions by R1 sgRNA-mediated HDR in pig embryos (Fig. 3B). Another insertion showed a precise addition at the 3′ end, whereas 117 bps indels were noted at the 5′ side of the modification site (Fig. 3C). In addition, we failed to detect HDR induced by F2 sgRNA or R1 sgRNA with 10 ng/μL ssODN (Table 2), suggesting that CRISPR/Cas9 and ssODN-mediated HDR were highly dependent on the targeting efficiency and the ssODN concentration.


Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Wang X, Zhou J, Cao C, Huang J, Hai T, Wang Y, Zheng Q, Zhang H, Qin G, Miao X, Wang H, Cao S, Zhou Q, Zhao J - Sci Rep (2015)

R1 sgRNA-induced site-specific insertion with ssODN through HDR.A.(A) schematic of the targeting site with the ssODN sequence used to introduce an exogenous KpnI sequence (underlined). The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from individual blastocyst digested with KpnI restriction enzymes. Three out of 23 blastocysts demonstrated site-specific KpnI sequence insertion through HDR. (C) Sequence analysis of the three blastocysts at the target site. Two blastocysts showed precise HDR-based addition of the KpnI sequence. One blastocyst showed precise addition at the 3′ end, and 117 bp indels were noted at the 5′ side of the modification site. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. KpnI sequence is underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4543986&req=5

f3: R1 sgRNA-induced site-specific insertion with ssODN through HDR.A.(A) schematic of the targeting site with the ssODN sequence used to introduce an exogenous KpnI sequence (underlined). The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. (B) RFLP agarose gel electrophoresis showing PCR product of target region derived from individual blastocyst digested with KpnI restriction enzymes. Three out of 23 blastocysts demonstrated site-specific KpnI sequence insertion through HDR. (C) Sequence analysis of the three blastocysts at the target site. Two blastocysts showed precise HDR-based addition of the KpnI sequence. One blastocyst showed precise addition at the 3′ end, and 117 bp indels were noted at the 5′ side of the modification site. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. KpnI sequence is underlined.
Mentions: The DSBs mediated by CRISPR/Cas9 can stimulate a homologous recombination in the presence of a DNA donor with the appropriate homology arms. Recent works have demonstrated that single-stranded DNA oligonucleotides (ssODNs) can be used as substitutes for conventional plasmid-based targeting vectors as donor templates for homology-directed repair (HDR)36. Because of its high mutagenesis efficiency, we hypothesized that R1 sgRNA could assist in HDR. Hence, we co-injected Cas9 mRNA, sgRNA and ssODN containing 6 bp KpnI restriction site flanked by 26 bps homologous sequences on each side into mature MII pig oocytes (Fig. 3A). After parthenogenetic activation, oocytes were cultured to the blastocyst stage and single blastocysts were picked for genotyping. In these experiments, the RFLP assays, as shown in Fig. 3B, identified 3 out of 23 R1 sgRNA-injected blastocysts carrying the KpnI site at the target locus, indicating R1 sgRNA yielded an HDR efficiency as high as 13.04% with ssODN at a concentration of 80 ng/μL. Subsequent sequence analyses indicated two precise KpnI site insertions, which demonstrated successful targeted restriction site insertions by R1 sgRNA-mediated HDR in pig embryos (Fig. 3B). Another insertion showed a precise addition at the 3′ end, whereas 117 bps indels were noted at the 5′ side of the modification site (Fig. 3C). In addition, we failed to detect HDR induced by F2 sgRNA or R1 sgRNA with 10 ng/μL ssODN (Table 2), suggesting that CRISPR/Cas9 and ssODN-mediated HDR were highly dependent on the targeting efficiency and the ssODN concentration.

Bottom Line: Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks.The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%.Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

No MeSH data available.