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, 291 (28), 14457-67

Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes

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Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes

Sean Chen et al. J Biol Chem.

Abstract

The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.

Keywords: CRISPR, Cas9, Genome Editing, Electroporation, RNP; gene knockout; genetics; mouse; ribonuclear protein (RNP); tyrosinase.

Figures

FIGURE 1.
FIGURE 1.
CRISPR-EZ efficiently generates NHEJ-mediated indel mutations in mouse embryos. A, diagram illustrating the workflow of the CRISPR-EZ technology in mouse genome editing. Fertilized embryos were combined with pre-assembled Cas9/sgRNA RNPs for electroporation and then transferred to pseudopregnant mothers to generate edited mice. B, gene schematic illustrating the NHEJ-mediated editing design that targets Tyr exon 1. The HinfI restriction site within Tyr exon 1, located 2 nt upstream of the PAM, will be disrupted upon successful NHEJ editing. Arrows indicate the positions of primers that amplify a DNA fragment for RFLP genotyping analyses. C and D, representative RFLP genotyping analyses (C) and sequencing confirmation (D) of Tyr NHEJ editing in mouse morula embryos using 8 μm Cas9/sgRNA RNPs under different electroporation conditions. C, presence of an undigested PCR product (200 bp) indicates successful NHEJ editing. Top, nested PCR amplicons from morula embryos following CRISPR-EZ; bottom, HinfI digestion using nested PCR amplicons in RFLP analyses. D, chromatograms and alignment of sequences from two edited mouse morula embryos compared with the wild type Tyr sequence. Red boxes indicate edited sequences. E and F, key experimental conditions in CRISPR-EZ were optimized to achieve high editing efficiency on Tyr and favorable embryo viability in culture. Three electroporation conditions and two Cas9 RNP concentrations were compared for Tyr NHEJ editing efficiency (E) and for embryo viability (F). F, percent survival was calculated as the ratio between the number of embryos that developed to the morula stage and the total number of zygotes electroporated. G, CRISPR-EZ achieved efficient NHEJ editing of Cdh1, Cdk8, and Kif11. Following CRISPR-EZ using optimized conditions (8 μm Cas9/sgRNA RNPs, two pulses of electroporation at 3-ms pulse length), the efficiency of NHEJ editing on Cdh1, Cdk8, and Kif11 was measured by RFLP analyses. H, sequence validation is shown for representative Cdh1, Cdk8, and Kif11 editing events.
FIGURE 2.
FIGURE 2.
CRISPR-EZ generates HDR-mediated genome modifications in mice. A, diagram illustrating the HDR editing scheme targeting Tyr exon 1. A synthesized 92-nt ssDNA donor oligo directs HDR-mediated editing, which replaces the endogenous HinfI restriction site with an EcoRI site, and causes a frameshift mutation to create a premature stop codon in Tyr exon 1. Arrows indicate the positions of primers that amplify a 200-bp DNA fragment for RFLP genotyping analyses. B, RFLP genotyping analyses revealed the successful NHEJ and HDR editing in morula embryos. Nested PCR amplicons (top row) were digested with both HinfI (middle row) and EcoRI (bottom row) to assay for NHEJ editing and HDR editing, respectively. HDR-specific digestion products (∼100 bp, migrate as one band) are marked with black arrowheads. C, representative Tyr-edited mouse litters for a microinjection experiment (left), a CRISPR-EZ experiment at 1-ms pulse length (middle), and a CRISPR-EZ experiment at 3-ms pulse length (right). D, quantification of coat color phenotypes of live Tyr-edited mice generated by microinjection and CRISPR-EZ experiments. E, CRISPR-EZ significantly improves mouse viability after genome editing compared with microinjection-based experiments. The University of California at Berkeley transgenic facility averages were calculated based on data collected across recent five CRISPR experiments that inject cas9 mRNA and sgRNAs for genome editing. F and G, albino mice obtained from CRISPR-EZ experiments were subjected to RFLP genotyping analyses (F) to demonstrate NHEJ and/or HDR editing, and select albino mice were sequence-confirmed (G). HDR-specific digestion products (∼100 bp, migrating as one band) are marked with black arrowheads (F). Red boxes indicate edited sequences, and red letters indicate the HDR-mediated precise modification (G). H, diagram illustrating the editing scheme to delete exon 3 of the Mecp2 gene by CRISPR-EZ. Two sgRNAs were designed to direct Cas9 cleavage in Mecp2 intron 2 and intron 3 to generate the ∼720-bp deletion of exon 3. Arrows indicate the positions of primers used for PCR genotyping that amplifies across the deleted region. I and J, representative PCR genotyping analyses (I) and sequencing confirmation (J) for assessing the editing efficiency of Mecp2 in mouse morula embryos. Red boxes indicate deleted sequences. K, diagram illustrating our HDR editing scheme that inserts a V5 epitope tag (42 bp in length) at the 3′ of the Sox2 open reading frame via a 162-nt ssDNA donor oligo using CRISPR-EZ. Arrows indicate the positions of primers that amplify a 180-bp DNA fragment across the edited genomic region. L and M, PCR genotyping analysis (L) and immunofluorescence staining (M) for assessing the editing efficiency of the sox2 gene in mouse morula and blastocyst embryos, respectively. Scale bar, 20 μm.
FIGURE 2.
FIGURE 2.
CRISPR-EZ generates HDR-mediated genome modifications in mice. A, diagram illustrating the HDR editing scheme targeting Tyr exon 1. A synthesized 92-nt ssDNA donor oligo directs HDR-mediated editing, which replaces the endogenous HinfI restriction site with an EcoRI site, and causes a frameshift mutation to create a premature stop codon in Tyr exon 1. Arrows indicate the positions of primers that amplify a 200-bp DNA fragment for RFLP genotyping analyses. B, RFLP genotyping analyses revealed the successful NHEJ and HDR editing in morula embryos. Nested PCR amplicons (top row) were digested with both HinfI (middle row) and EcoRI (bottom row) to assay for NHEJ editing and HDR editing, respectively. HDR-specific digestion products (∼100 bp, migrate as one band) are marked with black arrowheads. C, representative Tyr-edited mouse litters for a microinjection experiment (left), a CRISPR-EZ experiment at 1-ms pulse length (middle), and a CRISPR-EZ experiment at 3-ms pulse length (right). D, quantification of coat color phenotypes of live Tyr-edited mice generated by microinjection and CRISPR-EZ experiments. E, CRISPR-EZ significantly improves mouse viability after genome editing compared with microinjection-based experiments. The University of California at Berkeley transgenic facility averages were calculated based on data collected across recent five CRISPR experiments that inject cas9 mRNA and sgRNAs for genome editing. F and G, albino mice obtained from CRISPR-EZ experiments were subjected to RFLP genotyping analyses (F) to demonstrate NHEJ and/or HDR editing, and select albino mice were sequence-confirmed (G). HDR-specific digestion products (∼100 bp, migrating as one band) are marked with black arrowheads (F). Red boxes indicate edited sequences, and red letters indicate the HDR-mediated precise modification (G). H, diagram illustrating the editing scheme to delete exon 3 of the Mecp2 gene by CRISPR-EZ. Two sgRNAs were designed to direct Cas9 cleavage in Mecp2 intron 2 and intron 3 to generate the ∼720-bp deletion of exon 3. Arrows indicate the positions of primers used for PCR genotyping that amplifies across the deleted region. I and J, representative PCR genotyping analyses (I) and sequencing confirmation (J) for assessing the editing efficiency of Mecp2 in mouse morula embryos. Red boxes indicate deleted sequences. K, diagram illustrating our HDR editing scheme that inserts a V5 epitope tag (42 bp in length) at the 3′ of the Sox2 open reading frame via a 162-nt ssDNA donor oligo using CRISPR-EZ. Arrows indicate the positions of primers that amplify a 180-bp DNA fragment across the edited genomic region. L and M, PCR genotyping analysis (L) and immunofluorescence staining (M) for assessing the editing efficiency of the sox2 gene in mouse morula and blastocyst embryos, respectively. Scale bar, 20 μm.

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