Genome Modified Mice

Genome edited founder mice with precise deletions and insertions up to 4 kb using CRISPR/HiFi Cas9 are generally produced within 4-6 months at a 100% success rate, provided mice with the desired modification are viable and fertile. Each new project undergoes a project feasibility assessment so we can advise on an approach that gives you the best chance of success.

Transgenic mice are preferentially produced through VGER using piggyBac transgenesis. This strategy delivers transgenes up to 10 kb into the mouse genome efficiently, and larger constructs somewhat less efficiently, at single copies per site. piggyBac transposons usually land in areas of open chromatin within introns and intergenic DNA, reducing transgenic silencing concerns associated with random DNA integrations. We can map most random insertion sites and design genotyping assays to assess zygosity, simplifying breeding strategies.

VGER maintains stocks of validated, hyperactive piggyBac transposase mRNA and we advise on the production and/or sourcing of piggybac transposons.

CRISPR interference (CRISPRi) is an effective technology for transcriptional gene repression, non-coding RNA regulation, and regulatory element repression. We can efficiently produce mice genetically expressing one or more sgRNAs using piggyBac transgenesis. These new transgenic lines are then bred to global or conditional dCas9-KRAB expressing mice available through JAX and VGER to repress genes in either a global, conditional, or tissue-restricted manner. Moderate to strong gene repression has been reported by our collaborating investigators piloting this technology. We are happy to discuss whether this technology is right for you.

Gluckinase CRISPRi mouse model

Gck CRISPR interference (CRISPRi) results in post-natal lethality. (A) UCSC genome browser mm10 view of the Gck neuroendocrine-specific TSS. Two non-overlapping guides were positioned around 50 bp upstream of the TSS at a promotor-specific element required for Gck expression (PMID: 15365616). (B) Mice with a Gck-sgRNA expressing transposon were bred to mice expressing dCas9-KRAB. (C) Both WT and CRISPRi genotypes are present at expected ratios at P0-P1, whereas only one Gck CRISPRi pup in 28 was observed at 3 weeks of age (anticipated 75% WT and 25% CRISPRi). (D) Gck expression is reduced by more than 95% in Gck CRISPRi P0-P1 pups (n = 4 each) and in the single 3-week-old Gck CRISPRi mouse (n = 1 each). (E) The Gck CRISPRi 3-week-old pup is smaller and has higher blood glucose compared to its sex-matched litter and cage mate.

CRISPR screens are a powerful way to identify genes involved in cellular functions.  In vivo screens can identify genes involved in complex processes like organogenesis, tissue regeneration, oncogenesis, metastasis, and immune signaling. A major limitation of in vivo CRISPR screening is getting sgRNAs into cells. Viral sgRNA delivery methods do not work well for all tissues, it can be hard to get high mutation rates while also balancing the need to avoid disrupting multiple genes per cell. Finally viral libraries must be reproduced for each screen, adding additional complexity and variability to each analysis.

A method using transgenic sgRNA expression in mice coupled with standard Cre and Cas9-expressing alleles, demonstrates the feasibility of this approach for performing screens on a set number of targets: Large-scale multiplexed mosaic CRISPR perturbation in the whole organism – PubMed. VGER has adapted this technology to generate mice containing around 10 curated targets per array to be assessed using information-rich screening approaches, such as single-cell omics and spatial read-outs. The first models are currently undergoing experimental validation, but we have clear evidence that the arrays recombine properly, sgRNAs are active and generate CRISPR-mediated indels, and we can generate global KO mice by simple breeding strategies.

We envision this approach as particularly valuable for performing information-rich secondary screens on a subset of gene candidates obtained from methods such as whole genome screening, RNAseq data, or a candidate list from literature. Collecting a set of mouse models with global or conditional mutations in genes of interest is expensive. Producing a single sgRNA array model can be used for both screening and production of single mutant mice for any of the targets in the array for further study.

Advantages of in vivo screening with transgenic sgRNA expression:

• Induce array recombination in practically any tissue
• One guide RNA is expressed per cell
• Screen mutant cells head-to-head (mosaic analysis) in the same tissue
• Highly reproducible
• The mouse is a living, reusable sgRNA library
• Can breed out individual mutations from the original strain with a germline Cre