CRISPR Mice Generation Service Knock-In & Knock- Out Mice
We use our proprietary TARGATT™, Cas9-CRIS PR service, and homologous recombination technologies to make various forms of gene modification including: CRISPR knockout mice, CRISPR knockin mice, gene replacement, point mutation, and deletions.
The CRISPR/Cas9 system uses the Cas9 nuclease to facilitate RNA-guided site-specific DNA cleavage. The system consists of two components: (1) mammalian codon-optimized version of the Cas9 protein carrying a nuclear localization signal to ensure nuclear compartmentalization in mammalian cells; (2) guide RNAs (gRNAs) to direct Cas9 protein to sequence-specifically cleave the targeted DNA. The advantage of CRISPR/Cas9 over ZFNs or TALENs is its scalability and multiplexibility in that multiple sites within the mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing in mammalian cells.
CRISPR Knock-Out or Knock-in Mouse Generation Timeline
1. Targeting DNA Vector Creation
gRNA Design and Construction (2-4 gRNAs)
gRNA in vitro Functional Validation (2-4 gRNAs)
Donor DNA Construction
In vitro Trabscription and QC for Microinjection
A Report on cloning and validation
|2. CRISPR DNA Pronuclear Microinjection||1-2 months|
|3. Animal care, housing, and genotyping
Pups generated and genotyping showing the proof of precise gene insertion
|2-3 months||At least 1 founder
A final report on the project including the original targeting strategy and microinjection detail
Why choose Applied StemCell's Transgenic Mouse Services?
Applied StemCell is one of the first and most experienced provider of the CRISPR-Cas9 Technology. Our expert team of scientists have extensively researched and upgraded the CRISPR system to optimize the efficiency of Cas9 cutting, modification efficiency and even improve birth rate of mice. The findings were presented at the prestigious AACR Annual Meeting 2016 in New Orleans in April. Some of key findings have been noted under technical details.
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
FAQ for CRISPR-Cas9 Engineering of mouse models
1. What is the efficiency of CRISPR-Cas9 technology as compared to TARGATT™ and random transgenesis?
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%.
2. How many embryos are microinjected for validation of gRNAs?
We inject 50 embryos for in vivo validation of gRNAs.
3. What is the correlation between cutting in the embryo test (in vivo gRNA validation) and the final mouse?
The Cas9 cutting in the test embyros are 80-90% reflective in final pups born.
4. Which technology is suitable for inserting a gene of interest with GFP or Cre-ERT?
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.
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: Agarose gel electrophoresis of PCR results in F0 mice (#15, 19, and 26) with site-specific gene knockin. 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 the cytoplasm of C57BL/6 (B6) embryo; (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.
Applied StemCell's cited/published articles
- Hu, J. K., et al. (2016). PloS one, 11(5), e0156074
- Smalley, E. (2016) CRISPR mouse model boom, rat model renaissance. Nature Biotechnology. 34: 893-894
- Baker, Monya. (2014) Gene editing at CRISPR speed. Nature Biotechnology 32, 309–312.
CRISPR Knock-in H11 Locus in Pigs
- Ruan, J., et al. (2015). Scientific reports, 5:14253. doi: 10.1038/srep14253.
Knock-in, Knockout, Conditional Knock-out