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, 200 (2), 423-30

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

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

Wenning Qin et al. Genetics.

Abstract

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. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. 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. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

Keywords: CRISPR; Cas9; electroporation; mouse zygote.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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.
Figure 3
Figure 3
ZEN enables high throughput genome editing in mice. (A) Flow chart of CRISPR/Cas9-mediated genome editing delivered by ZEN. (B) ZEN compared to microinjection.

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