One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering

Abstract

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

Figure 1

One step generation of the Sox2-V5 allele. (A) Schematic of the Cas9/sgRNA/oligo targeting site at the Sox2 stop codon. The sgRNA coding sequence is underlined, capitalized, and labeled in red. The protospacer-adjacent motif (PAM) sequence is labeled in green. The stop codon of Sox2 is labeled in orange. The oligo contained 60bp homologies on both sides flanking the DSB. In the oligo donor sequence, the V5 tag sequence is labeled as a green box. PCR primers (SF, V5F, and SR) used for PCR genotyping are shown as red arrowheads. (B) Upper panel, PCR genotyping using primers V5F and SR produced bands with correct size in targeted ES samples T1 to T5, but not in WT sample. Lower panel, PCR genotyping using primers SF and SR produced slightly larger products, indicating the 42bp V5 tag sequence was integrated. T1 only contain larger product, suggesting either both alleles were targeted, or one allele failed to amplify. (C) PCR products using primers SF and SR were cloned into plasmid and sequenced. Sequence across the targeting region confirmed correct fusion of V5 tag to the last codon of Sox2. (D) Western blot analysis identified Sox2-V5 protein using V5 antibody in ES cells containing Sox2-V5 allele. Beta-actin was shown as the loading control. Because beta-actin and Sox2-V5 run at the same size, the samples for the V5 signal and beta-actin were run in parallel on the same gel. (E) Immunostaining of targeted blastocyst using V5 antibody showed signal in ICM. Scale bar, 50μm. (F) Immunostaining of targeted ES cells using V5 antibody showed uniform Sox2 expression. Scale bar, 100μm.

Figure 2

One step generation of an endogenous reporter allele. (A) Schematic overview of strategy to generate a Nanog-mCherry knock-in allele. The sgRNA coding sequence is underlined, capitalized, and labeled in red. The protospacer-adjacent motif (PAM) sequence is labeled in green. The stop codon of Nanog is labeled in orange. The homologous arms of the donor vector are indicated as HA-L (2kb) and HA-R (3kb). The restriction enzyme used for Southern blot analysis is shown, and the Southern blot probes are shown as red boxes. (B) Southern analysis of Nanog-mCherry targeted allele. NcoI-digested genomic DNA was hybridized with 3′external probe. Expected fragment size: WT (wild type) = 11.5 kb, T (targeted) = 5.6 kb. The blot was then stripped and hybridized with mCherry internal probe. Expected fragment size: WT = N/A, T = 6.6 kb. (C) Nanog-mCherry targeted blastocysts showed expression in ICM. Mouse ES cell lines derived from targeted blastocysts remain mCherry positive, and the mCherry expression disappear upon differentiation. Scale bar, 100μm. (D) Schematic overview of strategy to generate an Oct4-eGFP knock-in allele. The sgRNA coding sequence is underlined, capitalized, and labeled in red. The PAM sequence is labeled in green. The homologous arms of the donor vector are indicated as HA-L (4.5kb) and HA-R (2kb). The IRES-eGFP transgene is indicated as a green box, and the PGK-Neo cassette is indicated as a grey box. The restriction enzyme used for Southern blot analysis is shown, and the Southern blot probes are shown as red boxes. (E) Oct4-eGFP targeted blastocysts showed expression in ICM. Scale bar, 50μm. Mouse ES cell lines derived from targeted blastocysts remain GFP positive. Scale bar, 100μm. (F) Southern analysis of Oct4-eGFP targeted allele. Southern analysis of Oct4-eGFP targeted allele. HindIII-digested genomic DNA was hybridized with 3′external probe. Expected fragment size: WT = 9 kb, Targeted = 7.2 kb. The blot was then stripped and hybridized with eGFP internal probe. Expected fragment size: WT = N/A, Targeted = 7.2 kb. See also Figure S2.

Figure 3

One step generation of a Mecp2 floxed allele. (A) Schematic of the Cas9/sgRNA/oligo targeting sites in Mecp2 intron 2 and intron 3. The sgRNA coding sequence is underlined, capitalized, and labeled in red. The PAM sequence is labeled in green. In the oligo donor sequence, the loxP site is indicated as an orange box, and the restriction site sequences are in bold and capitalized. The oligo contained 60bp homologies on both sides flanking the DSB. Restriction enzymes used for RFLP and Southern blot analysis are shown, and the Southern blot probes are shown as red boxes. (B) Southern analysis of targeted alleles. Data of five mice are shown. EcoRI/NheI-digested genomic DNA was hybridized with the exon3 probe. Expected fragment size: WT = 5.2 kb, 2loxP = 0.7 kb, L2-loxP = 3.9kb, R1-loxP = 2kb. The blot was then stripped and hybridized with the exon4 probe. Expected fragment size: WT = 5.2 kb, 2loxP = 3.2 kb. L2-loxP = 3.9kb, R1-loxP = 3.2kb. The sequence of the floxed allele is shown in Fig. S4b. (C) In vitro Cre-mediated recombination of the floxed Mecp2 allele. The genomic DNA of targeted mice #1 and #3 was incubated with Cre recombinase, and used as PCR template. Primers DF and DR flanking the floxed allele produce shorter products upon Cre-dependent excision. Primers CF and CR detect the circular molecule, which only form upon Cre-loxP recombination. The position of each primer is shown at the bottom cartoon. The deletion and circular PCR products were sequenced and the sequences are shown in Fig. S4c. (D) Injection of Cas9 mRNA and both L2 and R1 sgRNA generated Mecp2 mutant allele with deletion of exon 3. PCR genotyping using primers DF and DR identified defined deletion events in mice #1, #6, and #8 (indicated by stars). (E) Sequences of three mutant alleles with exon 3 deletions in three mice. R2 and L1 sgRNA coding sequences were underlined, capitalized, and labeled in red. The PAM sequence is labeled in green. See also Figures S3 and S4.