One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering

Abstract

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

Figure 1. Multiplexed Gene Targeting in mouse ES cells

(A) Schematic of the Cas9/sgRNA-targeting sites in Tet1, 2, and 3. The sgRNA-targeting sequence is underlined, and the protospacer-adjacent motif (PAM) sequence is labeled in green. The restriction sites at the target regions are bold and capitalized. Restriction enzymes used for RFLP and Southern blot analysis are shown, and the Southern blot probes are shown as orange boxes. (B) Surveyor assay for Cas9-mediated cleavage at Tet1, 2, and 3 loci in ES cells. (C) Genotyping of triple-targeted ES cells, clones 51, 52, and 53 are shown. Upper: RFLP analysis. Tet1 PCR products were digested with SacI, Tet2 PCR products were digested with EcoRV, and Tet3 PCR products were digested with XhoI. Lower: Southern blot analysis. For the Tet1 locus, SacI digested genomic DNA was hybridized with a 5′ probe. Expected fragment size: WT = 5.8 kb, TM (targeted mutation) = 6.4 kb. For the Tet2 locus, SacI, and EcoRV double-digested genomic DNA was hybridized with a 3′ probe. Expected fragment size: WT = 4.3 kb, TM = 5.6 kb. For the Tet3 locus, BamHI and XhoI double-digested genomic DNA was hybridized with a 5′ probe. Expected fragment size: WT = 3.2 kb, TM = 8.1 kb. (D) The sequence of six mutant alleles in triple-targeted ES cell clone 14 and 41. PAM sequence is labeled in red. (E) Analysis of 5hmC levels in DNA isolated from triple-targeted ES cell clones by dot blot assay using anti-5hmC antibody. A previously characterized DKO clone derived using traditional method is used as a control. See also Figure S1.

Figure 2. Single- and Double-Gene Targeting In Vivo by Injection into Fertilized Eggs

(A) Genotyping of Tet1 single-targeted mice. (B) Upper: genotyping of Tet2 single-targeted mice. RFLP analysis; lower: Southern blot analysis. (C) The sequence of both alleles of targeted gene in Tet1 biallelic mutant mouse 2 and Tet2 biallelic mutant mouse 4. (D) Genotyping of Tet1/Tet2 double-mutant mice. Analysis of mice 1 to 12 is shown. Upper: RFLP analysis; lower: southern blot analysis. The Tet1 locus is displayed on the left and the Tet2 locus on the right. (E) The sequence of four mutant alleles from double-mutant mouse 9 and 10. PAM sequences are labeled in red. (F) Three-week-old double-mutant mice. All RFLP and Southern digestions and probes are the same as those used in Figure 1. See also Figures S2 and S3.

Figure 3. Multiplexed HDR-Mediated Genome Editing In Vivo

(A) Schematic of the oligo-targeting sites at Tet1 and Tet2 loci. The sgRNA-targeting sequence is underlined, and the PAM sequence is labeled in green. Oligo targeting each gene is shown under the target site, with 2 bp changes labeled in red. Restriction enzyme sites used for RFLP analysis are bold and capitalized. (B) RFLP analysis of double oligo injection mice with HDR-mediated targeting at the Tet1 and Tet2 loci. (C) The sequences of both alleles of Tet1 and Tet2 in mouse 5 and 7 show simultaneously HDR-mediated targeting at one allele or two alleles of each gene, and NHEJ-mediated disruption at the other alleles. See also Figure S4.

Figure 4. Mutiplexed Genome Editing in ES Cells and Mouse

(A) Multiple gene targeting in ES cells. (B) One-step generation of mice with multiple mutations. Upper: multiple targeted mutations with random indels introduced through NHEJ. Lower: multiple predefined mutations introduced through HDR-mediated repair.