Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

Abstract

Background: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs).

Results: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected.

Conclusions: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.

Keywords: CRISPR/Cas9; Conditional null allele; Genome editing; High-throughput production; Homology directed repair; Mouse models; Single-stranded DNA oligonucleotides.

Fig. 1

Designs for creating conditional alleles through CRISPR-mediated targeting with ssODN donor DNA. a Schematic for illustrating conditional targeting designs. Cas9 (gray) complexed with sgRNA (dark blue) binds to complementary DNA (blue) on the target strand after recognition of the PAM site (red). b Symmetrical design utilized two 60 bp homology arms, excluding the sgRNA target sequence and PAM site. The symmetrical ssODN donors were designed to be complementary to the target strand. c Asymmetrical design utilized a 36 bp PAM-distal and a 91 bp PAM-proximal homology arms [35]. The asymmetrical ssODN donors were designed to be complementary to the non-target strand. d Diagram illustrating the position of the loxP site insertion within the sgRNA target sequence in the ssODN donor. The loxP sequence was always inserted one base away from the Cas9 cut site, disrupting the sgRNA sequence in the ssODN donor and thereby preventing re-cutting by Cas9 after targeting

Fig. 2

Screening strategies for HDR and NHEJ alleles, and random ODN insertion. Relative positions of the primers and approximate sizes of PCR products are listed below each allele. Scissors represent target sites. a Genotyping schemes for detecting loxP donor sequences. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. b Genotyping for NHEJ events utilizes a three-primer system, with P1 being shared between P2 and P3. Primers P1 and P3 reside between 100 to 200 bp outside of the target site (an average deletion product size is depicted). The P1 + P3 primer pair may not always amplify a wild-type product, if the target sequences are too far apart. 1400 bp represents the average distance between loxP insertion sites. c Random ODN insertion PCR primers reside internal to homology arm sequence, and will amplify the expected size product if the ssODN donor has been incorporated elsewhere in the genome, away from the critical exon, in addition to the on-target locus. d Primers for the homology arm screen were used in a SYBR-green quantitative PCR reaction from DNA samples from the N1 generation, using β-actin as a two-copy normalization control

Fig. 3

Conditional KO ssODN (a) and lssDNA (b) targeting attempts. Each donut chart represents the summation of each allele type for all F0 mice genotyped, by ODN donor. In the center of the chart is the total number of F0 mice genotyped. Percentages of each allele type of the total number of mice genotyped are listed on each segment. 5′ and 3′ loxP: Includes animals genotyped for both 5′ and 3′ loxP sites, irrespective of the presence of any additional alleles (e.g., animals with 5′ loxP, 3′ loxP and a null allele detected); Null allele: Includes animals genotyped for a null allele, which may also have a single HDR and/or NHEJ indel event; Single HDR event: Includes animals genotyped for a single HDR event with or without additional indel events; NHEJ Indel event: Animals in which only indel alleles were observed. a Summary of genotypes identified from ssODN targeting attempts, and symmetric and asymmetric homology arm designs. b Summary of genotypes identified from four lssDNA targeting attempts

Fig. 4

Pearson correlations for 5′ and 3′ loxP sites. Data plotted based on number of F0 mice with an HDR event (x-axis) versus any evidence for a DSB generated at the respective sgRNA site, 5′ and 3′, such as a NHEJ indel, HDR event, or the formation of a null allele (y-axis)

Fig. 5

Designs for creating and genotyping conditional alleles through CRISPR-mediated targeting with lssDNA donors. a Schematic for illustrating lssDNA conditional targeting designs. Cas9 (gray) complexed with sgRNA (dark blue) binds to complementary DNA (blue) on the target strand after recognition of the PAM site (red). A double-stranded DNA template is purchased with 100–200 bp homology arms. The 5′ homology arm begins with a GGG and ends with the 5′ sgRNA cut site. The 3′ homology arm terminates at an appropriate cDNA primer. b Genotyping schemes for detecting floxed and null alleles. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. Genotyping for a null allele uses primers P1 and P3 from each loxP genotyping reaction, which can also detect a full-length wild-type product (not shown) due to the smaller distance between loxP sites than typical designs from ssODN targeting attempts

Fig. 6

a Copy number data from quantitative SYBR Green PCR assays for Abat, Kctd7, Slc2a12, and Uqcr10, using primers following the design introduced in Fig. 2d for both 5′ and 3′ loxP ssODN donors. All mice screened for each conditional null attempt were heterozygous conditional null mice from the N1 generation that were sequence confirmed for the conditional allele. Negative control samples were CRISPR-targeted N1 mice that were wild-type for the allele being analyzed. b, c Copy number data from TaqMan^® Copy Number assays for (b) Slc2a12 and Smc1a (paired ssODN conditional null targeting attempts) and (c) Eif2s2 and Cd44 (single lssDNA conditional null targeting attempts) using N1 progeny from a single founder. Genotypes are listed with the mouse ID. Animals with an asterisk to the right of the genotype were sequence-confirmed for the conditional null allele. Of note, Smc1a is located on the X chromosome, thereby males only have one copy of the gene. As in (a), negative control samples were CRISPR-targeted N1 mice that were wild-type for the allele being analyzed