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Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.

Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, Malhotra A, Geurts AM, Chen YG, Wang H - Genetics (2015)

Bottom Line: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering.Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput.Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.

No MeSH data available.


Related in: MedlinePlus

CRISPR/Cas9-mediated indel mutations in mouse embryos delivered by electroporation. (A) Genotyping of mouse embryos targeted at the Tet1 locus. Mouse embryos were electroporated with Cas9 mRNA (100 ng/μl) and sgRNA targeting the Tet1 locus (50 ng/μl), cultured to blastocyst stage of development, and RFLP analysis performed as shown in the top panel. PCR products are sequenced and those showing overlapping sequencing traces were cloned and individual clones sequenced. In the bottom half, mutant alleles are shown for embryos 1 and 10 (indicated by red star) from the group treated with AT for 10 sec. The SacI restriction site at the target region, used for RFLP analysis, is bold and underlined. The protospacer adjacent motif (PAM) sequence is colored in green. Mutated bases are labeled in red. (B) Genotyping of mouse embryos targeted at theTet2 locus. Mutant alleles identified from embryos 4, 6, and 8 (indicated by red star) are shown. Only one mutant allele was recovered from embryos 4 and 8. The EcoRV site located within the target sequence is bold and underlined and PAM sequence colored in green.
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fig1: CRISPR/Cas9-mediated indel mutations in mouse embryos delivered by electroporation. (A) Genotyping of mouse embryos targeted at the Tet1 locus. Mouse embryos were electroporated with Cas9 mRNA (100 ng/μl) and sgRNA targeting the Tet1 locus (50 ng/μl), cultured to blastocyst stage of development, and RFLP analysis performed as shown in the top panel. PCR products are sequenced and those showing overlapping sequencing traces were cloned and individual clones sequenced. In the bottom half, mutant alleles are shown for embryos 1 and 10 (indicated by red star) from the group treated with AT for 10 sec. The SacI restriction site at the target region, used for RFLP analysis, is bold and underlined. The protospacer adjacent motif (PAM) sequence is colored in green. Mutated bases are labeled in red. (B) Genotyping of mouse embryos targeted at theTet2 locus. Mutant alleles identified from embryos 4, 6, and 8 (indicated by red star) are shown. Only one mutant allele was recovered from embryos 4 and 8. The EcoRV site located within the target sequence is bold and underlined and PAM sequence colored in green.

Mentions: To test for the efficiency of electroporation to deliver the CRISPR/Cas9 components, we chose the Tet1 and Tet2 genes as our targets, targeting these two loci with sgRNAs as previously described (Wang et al. 2013) (Figure S1). Mouse zygotes isolated from the B6D2F2/J strain were treated with AT for 10 sec, deposited into 10 μl of Opti-MEM, and then mixed with 10 μl of Cas9 mRNA/Tet1 sgRNA reagent at 40/20 and 100/50 ng/μl final concentration. Zygotes bathed in the CRISPR/Cas9 reagents were electroporated in a cuvette at the settings as described above. The embryos were washed three times with M2 solution and cultured in vitro for 3.5 days until blastocysts developed. Blastocysts were harvested and PCR products encompassing the target site were amplified and examined by RFLP analysis using the SacI site, which is part of the target sequence in Tet1. When a mixture of Cas9 mRNA at 40 ng/μl and Tet1 sgRNA at 20 ng/μl was used, no mutation was detected among 10 embryos treated with AT or 8 embryos not treated with AT (data not shown). A mixture of Cas9 mRNA at 100 ng/μl and Tet1 sgRNA at 50 ng/μl yielded 3 of 15 AT-treated and 1 of 15 AT-untreated embryos with mutations destroying the SacI site in the Tet1 locus (Figure 1A and Figure S1). Electroporating Cas9 mRNA at 400 ng/μl and Tet2 sgRNA at 200 ng/μl into mouse zygotes under the same conditions yielded 4 of 9 AT-treated embryos that carry mutations disrupting the EcoRV site in the Tet2 locus, as indicated by the presence of PCR products resistant to EcoRV digestion (Figure 1B and Figure S1). All PCR products were sequenced and samples with overlapping sequencing traces were subcloned and individual alleles determined (Figure 1B). We conclude that both Tet1 and Tet2 loci can be efficiently targeted in mouse embryos using electroporation to deliver the CRISPR/Cas9 reagents.


Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.

Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, Malhotra A, Geurts AM, Chen YG, Wang H - Genetics (2015)

CRISPR/Cas9-mediated indel mutations in mouse embryos delivered by electroporation. (A) Genotyping of mouse embryos targeted at the Tet1 locus. Mouse embryos were electroporated with Cas9 mRNA (100 ng/μl) and sgRNA targeting the Tet1 locus (50 ng/μl), cultured to blastocyst stage of development, and RFLP analysis performed as shown in the top panel. PCR products are sequenced and those showing overlapping sequencing traces were cloned and individual clones sequenced. In the bottom half, mutant alleles are shown for embryos 1 and 10 (indicated by red star) from the group treated with AT for 10 sec. The SacI restriction site at the target region, used for RFLP analysis, is bold and underlined. The protospacer adjacent motif (PAM) sequence is colored in green. Mutated bases are labeled in red. (B) Genotyping of mouse embryos targeted at theTet2 locus. Mutant alleles identified from embryos 4, 6, and 8 (indicated by red star) are shown. Only one mutant allele was recovered from embryos 4 and 8. The EcoRV site located within the target sequence is bold and underlined and PAM sequence colored in green.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: CRISPR/Cas9-mediated indel mutations in mouse embryos delivered by electroporation. (A) Genotyping of mouse embryos targeted at the Tet1 locus. Mouse embryos were electroporated with Cas9 mRNA (100 ng/μl) and sgRNA targeting the Tet1 locus (50 ng/μl), cultured to blastocyst stage of development, and RFLP analysis performed as shown in the top panel. PCR products are sequenced and those showing overlapping sequencing traces were cloned and individual clones sequenced. In the bottom half, mutant alleles are shown for embryos 1 and 10 (indicated by red star) from the group treated with AT for 10 sec. The SacI restriction site at the target region, used for RFLP analysis, is bold and underlined. The protospacer adjacent motif (PAM) sequence is colored in green. Mutated bases are labeled in red. (B) Genotyping of mouse embryos targeted at theTet2 locus. Mutant alleles identified from embryos 4, 6, and 8 (indicated by red star) are shown. Only one mutant allele was recovered from embryos 4 and 8. The EcoRV site located within the target sequence is bold and underlined and PAM sequence colored in green.
Mentions: To test for the efficiency of electroporation to deliver the CRISPR/Cas9 components, we chose the Tet1 and Tet2 genes as our targets, targeting these two loci with sgRNAs as previously described (Wang et al. 2013) (Figure S1). Mouse zygotes isolated from the B6D2F2/J strain were treated with AT for 10 sec, deposited into 10 μl of Opti-MEM, and then mixed with 10 μl of Cas9 mRNA/Tet1 sgRNA reagent at 40/20 and 100/50 ng/μl final concentration. Zygotes bathed in the CRISPR/Cas9 reagents were electroporated in a cuvette at the settings as described above. The embryos were washed three times with M2 solution and cultured in vitro for 3.5 days until blastocysts developed. Blastocysts were harvested and PCR products encompassing the target site were amplified and examined by RFLP analysis using the SacI site, which is part of the target sequence in Tet1. When a mixture of Cas9 mRNA at 40 ng/μl and Tet1 sgRNA at 20 ng/μl was used, no mutation was detected among 10 embryos treated with AT or 8 embryos not treated with AT (data not shown). A mixture of Cas9 mRNA at 100 ng/μl and Tet1 sgRNA at 50 ng/μl yielded 3 of 15 AT-treated and 1 of 15 AT-untreated embryos with mutations destroying the SacI site in the Tet1 locus (Figure 1A and Figure S1). Electroporating Cas9 mRNA at 400 ng/μl and Tet2 sgRNA at 200 ng/μl into mouse zygotes under the same conditions yielded 4 of 9 AT-treated embryos that carry mutations disrupting the EcoRV site in the Tet2 locus, as indicated by the presence of PCR products resistant to EcoRV digestion (Figure 1B and Figure S1). All PCR products were sequenced and samples with overlapping sequencing traces were subcloned and individual alleles determined (Figure 1B). We conclude that both Tet1 and Tet2 loci can be efficiently targeted in mouse embryos using electroporation to deliver the CRISPR/Cas9 reagents.

Bottom Line: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering.Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput.Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.

No MeSH data available.


Related in: MedlinePlus