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

Abstract

BackgroundThe 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).ResultsUsing 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0529-0) contains supplementary material, which is available to authorized users.

Using CRISPR/Cas9 engineering to generate a mouse with a conditional knockout allele for the promyelocytic leukemia zinc finger transcription factor

“…Encoding more than 50% of murine Plzf protein, exon 2 of the Plzf gene was selected based on the targeting strategy previously reported by the Pandolfi group (Barna et al, ) and on the conditional knockout vector design detailed by the International Mouse Phenotyping Consortium/Knockout Mouse Project (IMPC/KOMP ( Zbtb16 tm405544a(L1L2_Bact_P) ; http://www.informatics.jax.org/marker/MGI: 103222)). Established methods were used to co‐microinject 100 ng/μL Cas9 mRNA, 20 ng/μL validated sgRNA (each) and 100 ng/μL of ssODNs (each) into the cytoplasm of 200 C57BL/6NJ embryos (Lanza et al, ). Following micro‐injection, zygotes were transferred into pseudopregnant ICR recipient females at approximately 25–32 zygotes per recipient.…”

“…Both C57BL/6NJ and ICR mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Established methods of genotyping potential founder mice were followed (Lanza et al, ). Also, Sanger sequencing of cloned LoxP sites from mouse genomic DNA described previously were followed in these studies (Lanza et al, ).…”

“…Flanking PCR primers designed to amplify 80–180 base pair amplicons are listed in Supporting Information Figure S4 with the location, sequence, and number of mismatches from the original sgRNA. Genomic DNA from F2 mice was prepared as previously described (Lanza et al, ) and assayed by High Resolution Melt Analysis using the MeltDoctor HRM Master Mix (ThermoFisher Scientific Inc., Waltham, MA (#4415440)) according to the manufacturer's protocol and run on the Applied Biosystems QuantStudio 7 Flex Real‐Time PCR System (ThermoFisher Scientific Inc.). At least two wild‐type samples were analyzed concurrently with the test samples.…”

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

“…More recently, novel methods were developed to generate and use longer ssDNAs (~1000 nt) as homology donors in vivo in mice (Miura et al, 2015;Quadros et al, 2017;Lanza et al, 2018). Our results show that longer ssDNA constructs with visible dominant markers integrate in the fly genome in vivo albeit with lower efficacy than double stranded homology donors with large homology arms (i.e.…”

Generation and Validation of Tissue‐Specific Knockout Strains for Toxicology Research

“…Mice with a conditional allele, in which a critical exon(s) is flanked by loxP sites oriented in the same direction (floxed), can be made by gene targeting in mouse embryonic stem (ES) cells or by CRISPR-Cas9 genome editing. CRISPR-Cas9 can be faster and less labor intensive, but there are a number of limitations, including varying efficiencies and unintended mutations (Kosicki, Tomberg, & Bradley, 2018;Lanza et al, 2018). A discussion of the CRISPR-Cas9 system is provided in Background Information.…”