CRISPR Mice Generation Service
Applied StemCell utilizes its in-licensed CRISPR/Cas9 technology to generate mouse models with precise gene modifications such as knockout, knock-in and point mutation models for a variety of research and preclinical applications.
Learn more about our CRISPR Mouse Model Generation Service.
- Most up-to-date CRISPR design strategies and protocols
- 100% target-site cutting efficiency using validated gRNA
- >99% success rate in generating final mouse model
- Wide variety of genome modifications available
- F1 breeding to confirm germline transmission
- Animal IP belongs to researchers
- Electrophysiology and behavioral assessments for your custom mouse models
|Catalog ID#||Product Name||Price|
Choosing the Right Genome Editing Technology for Your Mouse Models: CRISPR, TARGATT™ and Beyond... (February 2017)
Factors affecting genome editing in mouse models using CRISPR/Cas9
Three major factors influence the efficiency of CRISPR/Cas9 in mouse models: the concentrations of Cas9 mRNA, gRNA and donor DNA. Each of these factors are interdependent which in turn affects three other intedependent parameters: the nuclease-mediated cutting rate, gene modification efficiency and birth rate of the pups.
Increasing gRNA and Cas9 concentration improves cutting/editing efficiency but affects birth rate
With increasing gRNA and Cas9 mRNA concentrations, the cutting efficiency increases to almost 97% but birth rate decreases. Therefore, a balance between gRNA and Cas9 concentrations, and birth rate of mice is required for maintaining a reasonable number of embryos microinjected and in turn to optimize birthrate and number of founders born.
Optimal donor concentration is also necessary to maintain balance between gene editing efficiency and birth rate
With increasing donor DNA concentration, the gene editing efficiency of CRISPR system increases but birth rate decreases. An optimal donor concentration of 200 ng/µl is recommended for microinjection in mouse embryos.
Gene editing efficiency and birth rate is also affected by size of insert and length of the homologous regions when using single stranded oligo donor DNA
A technical limitation of ssODN is its length. Most commercial vendors can manufacture up to 200 bases of single stranded DNA molecule. Therefore, homologous regions for a ssODN can be shortenend if insert size gets bigger. The system is perfect for point mutation insertions and can accomodate small fragment inserts up to 100 bp.
Distance between 2 cutting sites must be > 3kb
In projects necessitating 2 modification sites such as generating a conditional knock-out model with 2 LoxP inserts, the distance between cutting sites should be atleast 3 kb for simultaneous modification to avoid deletions between the two cut sites due to efficient NHEJ repair. If the distance between sites is < 3 kb, sequential modification is recommended. The recommended distance between the two insertion sites should be > 6 kb for efficient simultaneous modifications and to limit undesired deletion events.
Summary of our technical discussion about CRISPR gene modification in mouse models
- Optimal conditions are necessary for high efficiency generating mouse models
- Cas9/gRNA/donor DNA concentration; insert size and homology arm length are crucial for balancing cutting rate, gene editing efficiency and birth rate
- The optimal distance between two cutting sites for simultaneous modification is >6 kb
Efficiency of random transgenesis is dependent on the species and the strain of animal, if any. In mice, depending on the strain of mouse being used, the efficiency can vary between 3-30%. The CRISPR efficiency can be very high if the parameters are optimized for the strain of mouse and type of modification required (such as knockout, knock-in, and conditional knockout). The efficiency of TARGATT™ insertion at preselected safe harbor docking sites (attP sites) averages around 20-30%.
We inject 50 embryos for in vivo validation of gRNAs.
The Cas9 cutting in the test embyros are 80-90% reflective in final pups born.
The choice of gene knock-in technology will depend on the promoter that the transgene will be expressed under. If the knock-in fragment is under control of an endogenous promoter, CRISPR/Cas9 methodology will be adopted. If the transgene expression cassette requires a specific promoter (Ex. CRE expressed under control of a tissue-specific promoter), TARGATT™ technology will be better suited for integrating the transgene.
Our affordable Mouse Model Service uses upgraded CRISPR/ Cas9 design strategies, highly optimized CRISPR protocols and validation methods to generate mouse models successfully with a fast turnaround and reduced cost. We have generated > 500 mouse models with very high efficiency and success rate.
Some examples of available genetic modifications in mice include but not limited to:
- Constitutive and conditional knockouts
- Small fragment insertions, point mutations
- Large fragment knock-in: locus specific or safe harbor locus
- Gene tagging/ reporter gene insertion
- Gene replacement, humanized models
- Gene fusion/ translocation
- Gene overexpression, inducible expression, promoter modifications
- Gene editing/ correction to model human diseases
Leverage our extensive scientific expertise in CRISPR/Cas9 and other complementary genome editing technologies such as our proprietary TARGATT™ site-specific knock-in technology and traditional homologous recombination methods in generating a mouse model specific unique to your research niche.
1. Targeting DNA Vector Creation
gRNA Design and Construction (2-4 gRNAs)
A Report on Cloning and Validation
2. CRISPR DNA Pronuclear Microinjection
3. Animal Care, Housing and Genotyping
Pups generated and genotyping showing the proof of precise gene insertion
At least 1 Founder
Case Study# 1: CRISPR Knock-in Model - Generation of site-specific 2 kb large fragment knock-in mouse using CRISPR/Cas9
Goal: To insert a 2 kb large fragment DNA (gene of interest) at “a specified locus” in the mouse genome using CRISPR/Cas9.
The project was designed using a well optimized protocol to generate the transgenic mice: (1) Cas9 mRNA and gRNA were produced by in vitro transcription; (2) donor vector was constructed by in-fusion method: the plasmid contained 1 kb long 5’ and 1 kb long 3’ homologous arms flanking the gene of interest (2kb); (3) the mixture of Cas9 mRNA, gRNA and donor vector was microinjected into fertilized eggs of C57BL/6j mouse background.
Using a panel of genotyping primer pairs, three out of 31 pups born after microinjection (#15, 19, and 26) were identified as founders (F0), with the gene of interest inserted at the desired locus.
Figure 1: Agarose gel electrophoresis of PCR results in F0 mice (#15, 19, and 26) with site-specific gene knock-in. The left part of the gel shows the 5’ junction fragment (2,191 bp), and the right part of the gel shows the 3’ junction fragment (2,557 bp). [wt: wildtype control; M: 1 kb DNA ladder].
Case Study# 2: CRISPR Knock-out Model - Generation of a site-specific ~ 600 bp CRISPR knock-out mouse model
Goal: To delete a 587 bp fragment from a specific site in C57Bl/6 mouse genome using CRISPR
To achieve the model, three procedures were implemented. In the first step, a mixture of active guide RNA molecules (gRNAs) and qualified Cas-9 mRNA was injected into the cytoplasm of C57BL/6 embryo. The second step was to screen new mice born from the microinjection using PCR. And the third step was to confirm the positive animals by sequencing the modified region in the desired mouse locus.
Figure: Mice# 4, 5, 6, 8 were identified as F1 germline transmitted animals carrying the ~ 600 bp deletion. The lower band was extracted, purified and sequenced. The sequence results showed that the deletion removed the entire exon and was identical to parental sequence (not shown).
Case Study# 3: CRISPR Knock-out Model - Deletion of a ~100 bp fragment from exon of gene of interest
Goal: To delete a ~100 bp fragment from the N-terminus of the gene of interest in C57BL/6 mouse genome using CRISPR.
The mouse model required a ~ 100 bp DNA fragment to be deleted from the N-terminal of the gene of interest in order to cause a frame shift in downstream gene sequence, thereby leading to loss of protein. For this, we followed a well optimized protocol involving 3 steps: (1) a mixture containing active guide RNA molecules (gRNA) and qualified Cas-9 mRNA was injected into C57BL/6 (B6) embryos; (2) new mice born from the microinjection were screened by PCR; (3) potential positive animals were confirmed for the accurate sequence alterations by sequencing purified PCR products.
Figure: PCR amplification of exon region of F0 mice. Five mice born from the embryos microinjected with CRISPR cocktail were subjected to PCR amplification using genotyping primers. Mouse#3 was shown to carry a deletion fragment which was purified and sequenced.
Figure: Sequence alignment of wild type B6 mouse and founder mouse #3 showing deletion of the exon in the coding region(dark frame).
Case Study# 4: CRISPR Knock-in Model - Site-specific knock-in of a 27 bp tag in mice using CRISPR/Cas9
Five (#2, 5, 6 13 and 17) out of nineteen pups, 26%, were genotyped by restriction enzyme digest and sequencing to confirm the correct knock-in on one of the alleles (heterozygotes). A sequence with desired knock-in generated a different cutting pattern compared to its wild type counterpart.
CRISPR Mouse/ Rat Models: Knock-in, Knockout, and Conditional Knockout
Smalley, E. (2016). CRISPR mouse model boom, rat model renaissance. Nature Biotechnology. 34, 893–894.
Baker, M. (2014). Gene editing at CRISPR speed. Nature biotechnology, 32(4), 309-313.
CRISPR Knock-in H11 Locus in Pigs
Ruan, J., Li, H., Xu, K., Wu, T., Wei, J., Zhou, R., ... & Chen-Tsai, R. Y. (2015). Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Scientific reports, 5, 14253.
Knock-in, Knockout, Conditional Knock-out
- Park, J., Jung, E., Lee, S. H., & Chung, W. S. (2020). CDC50A dependent phosphatidylserine exposure induces inhibitory post-synapse elimination by microglia. bioRxiv.
- Ramachandra Rao, S., Fliesler, S. J., Kotla, P., Nguyen, M. N., & Pittler, S. J. (2020). Lack of Overt Retinal Degeneration in a K42E Dhdds Knock-In Mouse Model of RP59. Cells, 9(4), 896.
- Beurg, M., Barlow, A., Furness, D. N., & Fettiplace, R. (2019). A Tmc1 mutation reduces calcium permeability and expression of mechanoelectrical transduction channels in cochlear hair cells. Proceedings of the National Academy of Sciences, 116(41), 20743-20749.
- Goldring, A. C., Beurg, M., & Fettiplace, R. (2019). The contribution of TMC1 to adaptation of mechanoelectrical transduction channels in cochlear outer hair cells. The Journal of physiology.
- Hwang, S., He, Y., Xiang, X., Seo, W., Kim, S. J., Ma, J., ... & Kunos, G. (2019). Interleukin‐22 ameliorates neutrophil‐driven nonalcoholic steatohepatitis through multiple targets. Hepatology https://doi.org/10.1002/hep.31031.
- Dumesic, P. A., Egan, D. F., Gut, P., Tran, M. T., Parisi, A., Chatterjee, N., ... & Dou, F. (2019). An Evolutionarily Conserved uORF Regulates PGC1α and Oxidative Metabolism in Mice, Flies, and Bluefin Tuna. Cell metabolism.
- Liang, T., Zhang, H., Xu, Q., Wang, S., Qin, C., & Lu, Y. (2019). Mutant Dentin Sialophosphoprotein Causes Dentinogenesis Imperfecta. Journal of dental research, 0022034519854029.
- Qian, W., Miner, C. A., Ingle, H., Platt, D. J., Baldridge, M. T., & Miner, J. J. (2019). A human STAT1 gain-of-function mutation impairs CD8+ T cell responses against gammaherpesvirus-68. Journal of virology, JVI-00307.
- Kweon, S. M., Chen, Y., Moon, E., Kvederaviciutė, K., Klimasauskas, S., & Feldman, D. E. (2019). An Adversarial DNA N6-Methyladenine-Sensor Network Preserves Polycomb Silencing. Molecular Cell. https://doi.org/10.1016/j.molcel.2019.03.018
- Deng, F., He, S., Cui, S., Shi, Y., Tan, Y., Li, Z., ... & Peng, L. (2018). A Molecular Targeted Immunotherapeutic Strategy for Ulcerative Colitis via Dual-Targeting Nanoparticles Delivering miR-146b to Intestinal Macrophages. Journal of Crohn's and Colitis.
- Jo, S., Fonseca, T. L., Bocco, B. M. D. C., Fernandes, G. W., McAninch, E. A., Bolin, A. P., ... & Németh, D. (2018). Type 2 deiodinase polymorphism causes ER stress and hypothyroidism in the brain. The Journal of Clinical Investigation.
- Langston, R. G., Rudenko, I. N., Kumaran, R., Hauser, D. N., Kaganovich, A., Ponce, L. B., ... & Beilina, A. (2018). Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2. Neurochemical research, 1-14.
- Amara, N., Tholen, M., & Bogyo, M. (2018). Chemical tools for selective activity profiling of endogenously expressed MMP-14 in multicellular models. ACS Chemical Biology. doi: 10.1021/acschembio.8b00562.
- Allocca, S., Ciano, M., Ciardulli, M. C., D’Ambrosio, C., Scaloni, A., Sarnataro, D., ... & Bonatti, S. (2018). An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population. International journal of molecular sciences, 19(7).
*Peng, L., Zhang, H., Hao, Y., Xu, F., Yang, J., Zhang, R., ... & Chen, C. (2016). Reprogramming macrophage orientation by microRNA 146b targeting transcription factor IRF5. EBioMedicine, 14, 83-96.
*Hu, J. K., Crampton, J. C., Locci, M., & Crotty, S. (2016). CRISPR-mediated Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ triple gene disruption reveals NKT cell defects but not T follicular helper cell defects. PloS one, 11(5), e0156074.
*Besschetnova, T. Y., Ichimura, T., Katebi, N., Croix, B. S., Bonventre, J. V., & Olsen, B. R. (2015). Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis. Matrix Biology, 42, 56-73.
*McKenzie, C. W., Craige, B., Kroeger, T. V., Finn, R., Wyatt, T. A., Sisson, J. H., ... & Lee, L. (2015). CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice. Molecular biology of the cell, 26(18), 3140-3149.
*Bishop, K. A., Harrington, A., Kouranova, E., Weinstein, E. J., Rosen, C. J., Cui, X., & Liaw, L. (2016). CRISPR/Cas9-mediated insertion of loxP sites in the mouse Dock7 gene provides an effective alternative to use of targeted embryonic stem cells. G3: Genes, Genomes, Genetics, 6(7), 2051-2061.
For more journal references, please visit our comprehensive list of citations and reference publications.