Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Abstract

Background: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.

Results: Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring.

Conclusions: Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Keywords: CRISPR ribonucleoproteins; CRISPR/Cas9; Conditional knockout; Cre-LoxP; Easi-CRISPR; Homology directed repair; Reporter and recombinase knock-in; long ssDNA donors.

Fig. 1

Generation of a floxed Pitx1 allele using Easi-CRISPR. a–d The Easi-CRISPR strategy. a The two parts of the CRISPR guideRNA (crRNA + tracrRNA) and Cas9 protein. Combining them generates a ctRNP complex. The term ctRNP used here was formerly known as a cloning-free CRISPR/Cas9 system [28]. b A long ssDNA donor derived from a floxed exon cassette (or knock-in cassette as in Fig. 3) is mixed with ctRNP(s) to obtain the final Easi-CRISPR reagent cocktail for zygote injection. c Injection of a floxed ssDNA donor with right and left ctRNPs into zygotes results in replacement of the target exon with the floxed exon. For targeted insertions (as in Fig. 3) only a single ctRNP is required. d Following microinjection of the Easi-CRISPR reagent cocktail, genotyping and sequencing are used to identify founders with correctly modified genomes. e, f The Pitx1 wild-type allele, the Pitx1 ssDNA donor designed to flox exon 2 and the final targeted allele. The lengths of ssDNA, homology arms, and the distance between the two LoxP sites are shown. f Three genotyping PCRs and the primer combinations for these are indicated (5′ LoxP PCR, 5′ F + 5′ R primers; 3′ LoxP PCR, 3′ F + 3′ R primers; and full-length PCR, 5′ F + 3′ R primers). g Genotyping gel images from the ears of G0 offspring. The expected sizes of PCR amplicons (wild type (wt) or floxed) are indicated to the left of the gels. h Genotype interpretations are summarized below the gel image (M monoallelic, P partial insertion, N no insertion). Animals 3, 5, 7, and 8 had both the 5′ and 3′ LoxP sites in cis, while animals 2, 4, 9, and 10 contained only one LoxP site, due to partial insertion of the ssDNA cassette

Fig. 2

Generation of floxed alleles for Ambra1, Col12a1, and Ubr5 using Easi-CRISPR. a–c The wild-type alleles, floxing ssDNA donors for targeting exons 4, 2, and 58 of Ambra1, Col12a1, and Ubr5, respectively, and the corresponding floxed alleles. The lengths of ssDNA, homology arms, and the distance between the two LoxP sites are shown. d–f The primer pairs and genotyping PCRs are indicated as in Fig. 1f. The floxed allele schematics show minor differences in primer locations for each gene with respect to target exon size and location. g–i Genotyping of G0 offspring. The expected sizes of PCR amplicons (wild type or floxed) are indicated to the left of the gels. j–l Genotype interpretations are summarized below the gel images (M monoallelic, B biallelic, N no insertion). j Interpretation of Ambra1 genotyping: animals 1, 2, 3, 5, 7, and 8 had both the 5′ and 3′ LoxP sites located in cis. Note that animals 4 and 6 also contain additional amplicons smaller than the expected size (shown by arrows), suggesting that they harbor deletions and/or are mosaic. The sequences of the deletion alleles were not determined. Animals 1 and 5 were bred to wild type and a CD4 Cre mouse line (Fig. 5; Additional file 1: Figure S4). k Interpretation of Col12a1 genotyping: animals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites located in cis, and they carried deletions in their second allele (shown by the arrows); animal 3 was biallelic for both the 5′ and 3′ LoxP sites. The lanes between the marker and the samples in the full-length PCR gel image (bottom panel) were cropped out because they belonged to another experiment. l Interpretation of Ubr5 genotyping: animals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites located in cis

Fig. 3

Fusion of P2A-FlpO to the 3′ end of Fgf8 using Easi-CRISPR. a How Easi-CRISPR is used to generate knock-in alleles. b The Fgf8 locus, ssDNA donor, and the resulting targeted insertion allele. c Genotyping of G0 offspring. Primer locations for 5′ and 3′ junction PCRs are shown, along with expected amplicon sizes. Founder 4 has a correctly targeted P2A-FlpO insertion, as indicated by the presence and size of both 5′ and 3′ junction amplicons. The gel on the right shows that PCR amplification of this founder’s DNA with primers flanking the Fgf8 insertion site produced only the mutant amplicon, indicating that it is a biallelic insertion. WT wild type, M 100-bp marker; kb 1-kb marker. d Sequencing of 5′ and 3′ junctions in founder 4. The guide RNA sequence (italics), along with the cut site, PAM sequence (in red), a few bases of flanking sequences (above) and sequence chromatograms showing correctly targeted 5′ and 3′ junctions are shown below

Fig. 4

Germ line transmission of founder alleles generated using Easi-CRISPR. a–f Genotyping of offspring from two founders each for the Pitx1 and Ambra1 conditional alleles (a, b) and one founder each of the Fgf8, Slc26a5, Mmp9, and Mmp13 knock-in alleles (c–f) showing germ line transmission from all of these founders. As expected, all the pups from the Fgf8 founder contain a targeted allele because the founder is biallelic (c)

Fig. 5

Easi-CRISPR alleles perform as intended. Conditional alleles show the expected pattern of Cre-mediated deletion. a Genotyping of lymphocyte DNA isolated from a litter produced by mating the Ambra1 floxed founder 1 (Fig. 2g, lane 1) with a CD4 Cre strain. Offspring carrying both the floxed and Cre alleles (first three lanes) show the expected PCR amplicons. wt wild-type control sample, M 100-base pair marker. b Sequencing of a deletion allele showing Cre recombination (see Additional file 1: Figure S5 for comparing this sequence with the floxed allele sequence). c FGF8-P2A-FlpO activates a FLP-dependent tdTomato reporter in inner hair cells. A surface preparation of the cochlear epithelium isolated from a P1-P2 Fgf8 ^P2A-FlpO/+ ;Rosa26 ^RC::RFLG/+ animal was stained with Alexa488-phalloidin (green). Native tdTomato fluorescence (red) is evident in most inner hair cells (i), but not in outer hair cells (o)