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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.


CRISPR/Cas9-mediated HDR mutation in live mice delivered by electroporation. (A) Top panel, schematic of the target sequence and donor oligonucleotide from mouse Tet2 locus. The protospacer sequence is underlined and PAM sequence colored in green. Oligonucleotide-directed 2-bp changes are colored in red. Lower panel, RFLP analysis of 11 mice from the group electroporated with Cas9 mRNA, sgRNA targeting the Tet2 locus, and donor oligonucleotide at 400/200/400 ng/μl as indicated at the bottom of the panel. The cleaved band from the introduced EcoRI site is indicated by a red arrow. (B) Sequencing traces of PCR products encompassing the Tet2 target region from four mutant mice presented in A (86EP1–4). The PAM sequence is underlined by a green bar and the wild-type EcoRV site, a black bar. Overlapping sequencing traces among the mutant mice indicate existence of more than one allele among these mice, as compared to the WT mouse. The positions of the start of mutations are indicated by black arrows. (C) PCR products from two mice, 86EP1 and 86EP4, were cloned and individual clones sequenced. Sequences from clones 86EP1-2 and 86EP4-4 are shown. Precise modification converting EcoRV (underlined by a black bar) to EcoRI (underlined by a red bar) site was confirmed in these two mice.
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fig2: CRISPR/Cas9-mediated HDR mutation in live mice delivered by electroporation. (A) Top panel, schematic of the target sequence and donor oligonucleotide from mouse Tet2 locus. The protospacer sequence is underlined and PAM sequence colored in green. Oligonucleotide-directed 2-bp changes are colored in red. Lower panel, RFLP analysis of 11 mice from the group electroporated with Cas9 mRNA, sgRNA targeting the Tet2 locus, and donor oligonucleotide at 400/200/400 ng/μl as indicated at the bottom of the panel. The cleaved band from the introduced EcoRI site is indicated by a red arrow. (B) Sequencing traces of PCR products encompassing the Tet2 target region from four mutant mice presented in A (86EP1–4). The PAM sequence is underlined by a green bar and the wild-type EcoRV site, a black bar. Overlapping sequencing traces among the mutant mice indicate existence of more than one allele among these mice, as compared to the WT mouse. The positions of the start of mutations are indicated by black arrows. (C) PCR products from two mice, 86EP1 and 86EP4, were cloned and individual clones sequenced. Sequences from clones 86EP1-2 and 86EP4-4 are shown. Precise modification converting EcoRV (underlined by a black bar) to EcoRI (underlined by a red bar) site was confirmed in these two mice.

Mentions: To address whether the ZEN method can generate mice with precise genome modifications, we coelectroporated Cas9 mRNA, sgRNA targeting Tet2, and a 126-nt donor oligonucleotide with DNA sequence flanking the target site and a 2-nt mutation converting the naturally occurring EcoRV site (GATATC) in the target sequence to an EcoRI site (GAATTC) and recovered live mice. To identify CRISPR/Cas9-mediated NHEJ and HDR mutations, we digested the 466-bp PCR product encompassing the target site with EcoRV and with EcoRI independently (Figure 2). Using Cas9 mRNA at 400 ng/μl, Tet2 sgRNA at 200 ng/μl, and single-stranded DNA (ssDNA) donor at 400 ng/μl, we achieved 100% efficiency for introduction of the NHEJ mutation (loss of the EcoRV site; 11 positive mice of 11 mice analyzed). In addition, we identified three founder mice carrying HDR alleles as evident by acquisition of the EcoRI site (Figure 2, samples 86EP1, 86EP4, and 86EP11). We cloned the PCR products from 86EP1 and 86EP4, determined their DNA sequences, and confirmed that both mice acquired the EcoRI site (Figure 2C). In addition to this HDR allele, two other indel alleles were present in mouse 86EP1 and one indel allele in 86EP4, while no wild-type allele was found. Similar to mutant mice generated by microinjection, mice derived using the ZEN method often are mosaic. Using the TIDE software (Brinkman et al. 2014) to decompose sequencing traces, we found four mice carrying more than two Tet2 alleles among the 11 shown in Figure 2. Using a higher concentration of the donor oligonucleotide (1000 ng/μl), we obtained similar results with >70% NHEJ efficiency and >30% HDR efficiency at the Tet2 locus (Table S3).


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 HDR mutation in live mice delivered by electroporation. (A) Top panel, schematic of the target sequence and donor oligonucleotide from mouse Tet2 locus. The protospacer sequence is underlined and PAM sequence colored in green. Oligonucleotide-directed 2-bp changes are colored in red. Lower panel, RFLP analysis of 11 mice from the group electroporated with Cas9 mRNA, sgRNA targeting the Tet2 locus, and donor oligonucleotide at 400/200/400 ng/μl as indicated at the bottom of the panel. The cleaved band from the introduced EcoRI site is indicated by a red arrow. (B) Sequencing traces of PCR products encompassing the Tet2 target region from four mutant mice presented in A (86EP1–4). The PAM sequence is underlined by a green bar and the wild-type EcoRV site, a black bar. Overlapping sequencing traces among the mutant mice indicate existence of more than one allele among these mice, as compared to the WT mouse. The positions of the start of mutations are indicated by black arrows. (C) PCR products from two mice, 86EP1 and 86EP4, were cloned and individual clones sequenced. Sequences from clones 86EP1-2 and 86EP4-4 are shown. Precise modification converting EcoRV (underlined by a black bar) to EcoRI (underlined by a red bar) site was confirmed in these two mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: CRISPR/Cas9-mediated HDR mutation in live mice delivered by electroporation. (A) Top panel, schematic of the target sequence and donor oligonucleotide from mouse Tet2 locus. The protospacer sequence is underlined and PAM sequence colored in green. Oligonucleotide-directed 2-bp changes are colored in red. Lower panel, RFLP analysis of 11 mice from the group electroporated with Cas9 mRNA, sgRNA targeting the Tet2 locus, and donor oligonucleotide at 400/200/400 ng/μl as indicated at the bottom of the panel. The cleaved band from the introduced EcoRI site is indicated by a red arrow. (B) Sequencing traces of PCR products encompassing the Tet2 target region from four mutant mice presented in A (86EP1–4). The PAM sequence is underlined by a green bar and the wild-type EcoRV site, a black bar. Overlapping sequencing traces among the mutant mice indicate existence of more than one allele among these mice, as compared to the WT mouse. The positions of the start of mutations are indicated by black arrows. (C) PCR products from two mice, 86EP1 and 86EP4, were cloned and individual clones sequenced. Sequences from clones 86EP1-2 and 86EP4-4 are shown. Precise modification converting EcoRV (underlined by a black bar) to EcoRI (underlined by a red bar) site was confirmed in these two mice.
Mentions: To address whether the ZEN method can generate mice with precise genome modifications, we coelectroporated Cas9 mRNA, sgRNA targeting Tet2, and a 126-nt donor oligonucleotide with DNA sequence flanking the target site and a 2-nt mutation converting the naturally occurring EcoRV site (GATATC) in the target sequence to an EcoRI site (GAATTC) and recovered live mice. To identify CRISPR/Cas9-mediated NHEJ and HDR mutations, we digested the 466-bp PCR product encompassing the target site with EcoRV and with EcoRI independently (Figure 2). Using Cas9 mRNA at 400 ng/μl, Tet2 sgRNA at 200 ng/μl, and single-stranded DNA (ssDNA) donor at 400 ng/μl, we achieved 100% efficiency for introduction of the NHEJ mutation (loss of the EcoRV site; 11 positive mice of 11 mice analyzed). In addition, we identified three founder mice carrying HDR alleles as evident by acquisition of the EcoRI site (Figure 2, samples 86EP1, 86EP4, and 86EP11). We cloned the PCR products from 86EP1 and 86EP4, determined their DNA sequences, and confirmed that both mice acquired the EcoRI site (Figure 2C). In addition to this HDR allele, two other indel alleles were present in mouse 86EP1 and one indel allele in 86EP4, while no wild-type allele was found. Similar to mutant mice generated by microinjection, mice derived using the ZEN method often are mosaic. Using the TIDE software (Brinkman et al. 2014) to decompose sequencing traces, we found four mice carrying more than two Tet2 alleles among the 11 shown in Figure 2. Using a higher concentration of the donor oligonucleotide (1000 ng/μl), we obtained similar results with >70% NHEJ efficiency and >30% HDR efficiency at the Tet2 locus (Table S3).

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.