Generation of a conditional knock-out allele

Production of F 0 animals

Proof of principle for the RGEN-aided generation of conditional alleles employing two CRISPR/Cas9 cuts and two separate ssODN templates as donors was published in the early days of CRISPR/Cas9-aided mutagenesis [3]. However, the use of this strategy for allele generation has not flourished in the literature in the same way as other CRISPR-directed mutagenesis applications [18]. This is most likely because its success requires two concurrent events of homology-directed recombination occurring on the same allele, which remain less frequent than non-homologous end joining (NHEJ) events [5]; this is in keeping with our own experience of the approach (see examples below). We therefore decided to pilot the use of lssDNAs as a possible alternative to ssODN donors.

As a first test case, we aimed to generate a conditional allele in Syt7 by flanking the critical exon ENSMUSE00000225700 with loxP sites (Fig. 1a). This exon was chosen as defined by Skarnes and colleagues [19]. Specifically, the exon is common to the majority of coding transcripts in the gene, and its ablation results in frame-shift transcripts. Two pairs of sgRNAs were designed, centred on each of the genomic sequences to be interrupted by loxP (Fig. 1a), and synthesized to enhance the likelihood of simultaneous cuts on both sides of the same allele. A lssDNA donor corresponding to the floxed allele was generated as per Miura and colleagues ([10], and see Methods). Specifically, a double-stranded DNA template including a T7 transcription promoter followed by the 1149 bp sequence of the donor was obtained commercially (gBlock®, Integrated DNA Technologies (IDT); Fig. 1). A lssDNA was synthesized by in vitro transcription (IVT) and reverse transcription (detailed in Methods). The sgRNAs and lssDNA (the sequences are provided in Additional file 1: Table S1) were co-injected with Cas9 mRNA into one-cell embryos. One hundred thirty-eight injected embryos were re-implanted in pseudopregnant females. Seventeen pups were weaned and ear biopsies taken for screening of new alleles (the numbers are summarized in Additional file 1: Table S2, Syt7).

Fig. 1 Generation of a Syt7 floxed allele. a Diagrammatic representation of the genomic sequence with the Syt7 critical exon highlighted, the corresponding template for lssDNA synthesis and the position of sgRNAs for in vivo delivery together with the primer locations used for reverse transcription and for genotyping. Note loxP sites in the lssDNA prevent reprocessing of repaired alleles by CRISPR-Cas9 complex. Diagram shows the process for the generation of lssDNA through in vitro transcription and reverse transcription. HA homology arm. b PCR products amplified from genomic DNA extracted from the 17 F 0 born from the microinjection session using Syt7-F1 and Syt7-R1 primers. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). Sequence trace data derived from animals Syt7-4 and Syt7-8 are displayed in Additional file 2: Figure S1. Full size image

Table 1 Generation of conditional knock-out mice using lssDNA Full size table

Screening of F 0 generation and genotyping of F 1 animals

As animals of the F 0 generation were likely to be mosaic, we analyzed them by screening for the presence of the allele of interest [13]. Polymerase chain reaction (PCR) amplicons were produced from genomic DNA with primers flanking the homology arms and external to the donor (Syt7 primers R1 and F1, Fig. 1a). Their analysis on agarose showed two founders (Fig. 1b, Animals Syt7-1 and Syt7-6) containing deletions. The PCR products from founder animals were purified and sequenced by Sanger sequencing. The sequencing showed that a total of 10 animals out of 17 were mutated on target (Syt7, Table 1). Among them, five pups had indels at either or both 5′ and 3′ guide target sites. Three other animals (Syt7-1, Syt7-6 and Syt7-9) carried alleles with deletions of the sequence flanked by the two pairs of sgRNAs corresponding to non-cKO alleles. The remaining two mutants (Syt7-4 and Syt7-8) were carriers of the designed cKO allele, with sequencing traces suggesting Syt7-8 to be homozygous and Syt7-4 compound heterozygous with one cKO allele and one allele including the 3′ loxP and an indel in 5′ (Additional file 2: Figure S1).

Positive founders Syt7-4 and Syt7-8 were mated to wild-type (WT) animals, and the progeny (F 1 ) were analyzed. In contrast to the analysis of mosaic F 0 animals, sequencing of PCR fragments amplified from F 1 individuals allowed for definitive characterization of the edited alleles [13]. The outcome of the analysis of F 1 animals by PCR and sequencing, employing the same primers used for screening F 0 animals, is summarized in Table 2. Sequencing showed successful transmission of the correctly mutated sequence (cKO allele) by both founders to their progeny (individuals Syt7-4.1d and Syt7-8.1c, e, f and g).

Table 2 Characterization of animals for the generation of a Syt7 conditional allele Full size table

Screening of mutants obtained by co-injection of transcription activator-like effector nuclease (TALEN) and ssODNs showed that random integration of ssODNs can occur when using such a mutagenesis approach [20], illustrating the requirement of further validation of positive animals by a method allowing copy counting. We therefore checked for the presence of additional copies of the lssDNA donor sequence in the genome of F 0 and F 1 animals using digital droplet PCR (ddPCR) and a TaqMan™ assay centred on the critical exon present in the donor sequence run against a known two-copy reference assay (Syt7 exon 7, Dot1l reference assay, as per [13]). Table 2 shows the copy number of the donor sequence in each individual, illustrating the presence of additional copies in some F 0 (Syt7-8) and F 1 individuals (Syt7-8.1c, d, g and h).

In particular, copy counting for founder Syt7-8 (which was suggested as a potential homozygous for the cKO allele by PCR and sequencing) also revealed additional integrations of the lssDNA donor (close to 2.8 copies per genome, Table 2). The copy number obtained in the founder is not a clear integer number, which is not impossible in a mosaic animal. Analysis of the F 1 progeny confirmed the presence of an additional integration (Syt7-8.1c, d, g and h) and strongly suggested that this event was not physically linked to the targeted allele in the founder, as this integration could be segregated from the mutated allele in other F 1 progeny (Syt7-8.1e and f).

Copy counting of the critical exon also confirmed deletions of the target region in some F 0 (Syt7-4) and F 1 individuals (Syt7-4.1a, b and c; Syt7-8.1a). The ddPCR analysis also showed a reduced copy number of exon 7 in F 1 animals initially thought to be WT as an exon deletion had not been detected by standard PCR with external primers (Syt7-4.1a, b and c; Syt7-8.1a) Table 2. This suggests that these animals were bearing a deletion larger than the segments flanked by the genotyping primers.

In summary, the delivery of lssDNA donor together with CRISPR/Cas9 reagent to a modest number of one-cell embryos produced mosaic animals that transmitted a conditional allele. Some of the transmitting progeny were excluded upon further validation steps due to additional integrations of donor sequence.

Other conditional alleles

Production of F 0 animals

The pilot was next extended to include a further eight genes with the same design principles (Table 1 and Additional file 1: Table S2): Two sgRNAs were selected on each side of a critical exon in the genomic sequences to be interrupted by the loxP sites (details of sequences are given in Additional file 1: Table S1, designs in Additional file 4: Figure S3). Refining our strategy in the process of extending the pilot, we introduced standard sequences flanking the loxP sites in the designs, thus allowing us to re-use established diagnostic tests for the validation of alleles (restriction enzyme sites or LoxP-F and LoxP-R primers in Additional file 4: Figure S3). This facilitated the analysis of animals. CRISPR/Cas9 reagents and lssDNA were delivered to C57BL/6NTac one-cell embryos by pronuclear injection.

Screening of F 0 generation and genotyping of F 1 animals

F 0 and F 1 animals were analyzed according to the same strategy as that used for the Syt7 conditional allele: PCR using primers external to the donor homology arms (or two PCRs bridging the homology arms, depending on PCR efficiency) and a PCR amplifying the region flanked by the two loxP sites, all of which were analyzed by Sanger sequencing (Additional file 5: Figure S4, Additional file 6: Figure S5, Additional file 7: Figure S6, Additional file 8: Figure S7, Additional file 9: Figure S8, Additional file 10: Figure S9, Additional file 11: Figure S10 and Additional file 12: Figure S11). A total of 279 F 0 animals were analyzed, and 129 animals were identified as bearing mutations. Seven out of nine projects yielded founders bearing the conditional allele, with an additional one yielding a floxed allele with an unwanted point mutation. One project (Rapgef5) only yielded one founder bearing a conditional allele, that died before mating age. Correct conditional alleles were transmitted to the F 1 generation for four out of the seven projects where founder progeny were analyzed (Table 1). However, in at least three out of nine projects, other alleles were detected which contained unexpected point mutations identified at the F 0 generation (Inpp5k project, Additional file 12: Figure S11h; 6430573F11Rik project, Additional file 13: Figure S12a; Cx3cl1 project, Additional file 13: Figure S12b and c).

It is also noteworthy that illegitimate repairs [7] or partial integration(s) of the donor were detected frequently (in eight out of nine projects analyzed, see example in (Additional file 12: Figure S11d), highlighting the requirement of extensive allele validation by PCR and sequencing. These events—point mutations, partial and/or rearranged integrations—are reported as illegitimate repairs in Table 1.

Interestingly, F 0 animals with exon deletions were generated in all but one project as a by-product. Whenever null animals were required for ongoing research, these founders were also mated (numbers in brackets, Table 1). So far, germline transmission (GLT) of this additional allele was obtained in five out of six projects where positive founders were bred.

It is noteworthy that two out of these nine projects (Ikzf2 and Usp45) had been previously attempted employing ssODNs or plasmids without yielding founders with conditional alleles, in contrast to subsequent attempts with lssDNA donors (Additional file 1: Table S3).

F 0 and F 1 animals containing the cKO alleles were further validated by copy counting with a TaqMan™ assay centred on the floxed region. Importantly, copy counting of the floxed region in combination with the outcome of the targeted allele validation showed additional integrations in four out of seven projects analyzed (Table 1).

Point mutations remote from active sgRNA cutting site

Production of F 0 animals

Finally, we assessed whether the production of a point mutation distal from an active sgRNA cutting site, the generation of which has so far been unsuccessful by repeated attempts using other methods, could also be facilitated by the use of lssDNA. The first target for this pilot was the generation of the GckrP446L point mutation in C57BL/6NTac mouse embryos (sequence change illustrated in Additional file 15: Figure S14). We initially designed a strategy according to the standard approach, employing a ssODN and one efficient and specific sgRNA cutting as close as possible to the targeted nucleotide. However, some factors limited options for design, such as the close proximity of the target to the exon-intron junction and splice sites that should not be altered. Furthermore, the poor specificity of the target sequence (sequence conserved and repeated at two additional locations in the mouse genome; GRCm38.p5:10:82265447–82265469/12:21568953–21568975) rendered many guides unspecific. The closest sgRNA to the target nucleotide (sgRNA_20 (Fig. 2a)) was shown to be inactive by a Guide-it™ assay, where the CRISPR/Cas9 nuclease activity is assessed on a target DNA fragment in vitro (Fig. 3). This was subsequently confirmed by the fact that no mutagenesis was detected in microinjection session 1 where this sgRNA was used. Therefore, the closest efficient (as confirmed by Guide-it™ assay) and specific sgRNA that could be selected was cutting 34 nt away from the targeted base pair (sgRNA_3, Figs. 2a and 3). Thus, our next strategy employed sgRNA_3 and a ssODN donor, although a distance larger than 30 bp between the target sequence and the cutting site of the sgRNA can represent a barrier to the generation of a specific point mutation [9]. In addition to the targeted nucleotide mutation, a silent mutation was included in the ssODN donor template in order to abolish the protospacer adjacent motif (PAM) of the selected sgRNA and prevent re-processing of the mutated allele by the CRISPR/Cas9 system (Fig. 2a). The sgRNA activities were checked in vitro (Fig. 3), and each RNA was co-injected with Cas9 mRNA and the ssODN, as per the designs shown in Fig. 2a and Additional file 1: Table S1.

Fig. 2 GckrP446L point mutation. Different designs of reagents for genome editing employing (a) oligonucleotides or (b) a lssDNA donor. Donors were designed containing both coding (in red) and silent mutations (in black) that prevent reprocessing of engineered alleles in accordance with the selected sgRNAs. Guide sequences are named sgRNAs. The shared colour coding of guides and donors highlights reagents injected within the same mix Full size image

Fig. 3 Guide-it validation of the five sgRNAs synthesized for the generation of the GckrP446L point mutation. Cas9 protein is complexed with each sgRNA (B, D–G) and incubated with short double-stranded DNA fragments containing the protospacer target. Lanes A and C are controls and show the target template but no Cas9/sgRNA complex. The reactions are analyzed for cleavage by electrophoresis on agarose gel. L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). Protospacer sequences are detailed in Additional file 1: Table S1 Full size image

We anticipated that generating the desired mutation would be challenging, as the target base is a sub-optimal 34 base pairs away from sgRNA_3’s cut site. We therefore performed multiple injection sessions with two different ssODN designs (Gckrdonor_2 and Gckrdonor_3, centred or offset towards the targeted mutation, respectively; sequences in Additional file 1: Table S1) to enhance the likelihood of obtaining the desired point mutation. The outcome of these microinjections was analyzed by PCR and sequencing of the region of interest in a total of 90 pups and is summarized in Table 3. Although the silent mutation was detected in F 0 animals on five occasions, it was not accompanied by the mutation of interest (Table 3 and example in Fig. 4a, ssO-GckrP446L-54). Sequencing data from founders are shown in Additional file 16.

Table 3 Generation of a GckrP446L point mutation Full size table

Fig. 4 Screening by Sanger sequencing of animals for the generation of the GckrP446L point mutation with (a) oligonucleotides (F 0 individual ssO-GckrP446L-54) or (b) lssDNA donors (F 0 individuals lss-GckrP446L-11 and lss-GckrP446L-10 and F 1 individual lss-GckrP446L-11.1f). The figure shows Sanger sequencing chromatograms of an amplicon generated with primers anchored external to the intended site of donor sequence integration as detailed in Additional file 15: Figure S14. a ssODN donors only yielded introduction of the intended silent mutations, while (b) lssDNA yielded the desired mutation in some individuals (F 0 11 transmitting to 11.f) and only the silent mutations in others (F 0 10). Note that founders appeared homozygous (ssO-GckrP446L-54, lss-GckrP446L-11 and lss-GckrP446L-10) when analyzed by Sanger sequencing, but also could contain deletion alleles in trans, as suggested by copy counting (lss-GckrP446L-11 in Table 4). A summary of the microinjection session outcomes is detailed in Table 3, and raw sequencing data are provided in Additional file 16 Full size image

We subsequently designed an alternative strategy employing a larger (339 bases) lssDNA sequence and two sgRNAs flanking the region containing the targeted nucleotide. The sgRNAs were selected to introduce double-stranded breaks on each side of the target (40 and 98 nt away in 5′ and 3′, respectively), and their activity was checked in vitro. We consequently selected sgRNA_5.2 and sgRNA_3.1 as they were shown to be most active in vitro (Figs. 2b and 3). The donor sequence was designed with 100 nt homology arms flanking the cut sites, silent mutations that modify the seed sequences of the selected sgRNAs to prevent re-processing and the targeted base change (Fig. 2b). The lssDNA was synthesized in accordance with prior experiments and co-injected with Cas9 mRNA and the two sgRNAs in a single session, the outcome of which is shown in Table 3. Twenty-two pups were weaned, and ear biopsies were taken to screen for new alleles.

Screening of F 0 generation and genotyping of F 1 animals

Primers were designed in genomic regions flanking, but external to, the donor sequence to span the donor integration (GckrP446L-F2 and GckrP446L-R2 primers, Additional file 1: Table S1 and Fig. 2b). PCR amplicons were synthesized from genomic DNA and sequenced by Sanger sequencing. Sequencing data from all founders are shown in Additional file 16.

Sequencing showed that 14 animals out of 22 were mutated on target. Among them, eight individuals carried the designed knock-in (KI) allele (Table 3), with sequencing traces suggesting that four animals were homozygous for the KI (Fig. 4b). Three other individuals showed illegitimately repaired alleles (Table 3 and silent mutation only Fig. 4b).

Two of the four apparently homozygous positive F 0 s (lss-GckrP446L-11, lss-GckrP446L-19) were mated to WT animals for GLT of the mutated allele. The analysis of F 1 animals (summarized in Table 4) showed the successful transmission of the correctly mutated sequence by both founders (i.e. lss-GckrP446L-11.1f, Fig. 4b).

Table 4 Analysis of the GckrP446L project Full size table

Further model validation

We also checked for the presence of additional copies of the donor sequence in the genome of F 0 and F 1 animals using ddPCR and a TaqMan™ assay centred on the donor sequence (as per [13]). Table 4 shows the copy number of the donor sequence in each individual, illustrating a deletion likely spanning a fragment larger than the segments flanked by the genotyping primers (individuals lss-GckrP446L-11.1a, b, d, e and h, Table 4). Although both founders appeared homozygous for the point mutation by Sanger sequencing, lss-GckrP446L-11 also transmitted a deletion allele to its progeny, confirming mosaicism in this individual.

We next attempted to employ lssDNA donors for the generation of a mouse line bearing a point mutation in the Rims1 gene, which also had not been achieved with standard ssODN donors (Additional file 17: Figure S15 and Additional file 18: Figure S16; Additional file 1: Table S4, 1 positive founder/155 animals born (0.6%); this founder did not yield GLT, Additional file 1: Table S5). The new design employing lssDNA (Additional file 17: Figure S15) yielded founders bearing the correct mutation at a much higher frequency (4 positive founders/39 animals born (10%) with lssDNA donors), one of which achieved GLT of this second challenging point mutation (Additional file 1: Tables S4 and S5; Additional file 19: Figure S17; sequencing data in Additional file 20). Sequencing data from all founders for the point mutation (with ssODNs and lssDNA donors) are shown in Additional file 20.