CRISPR Knock-In, Knock-Out, PrimeCKO™ Conditional Knock-Out Rat
Applied StemCell utilizes CRISPR-Cas9 service to generate rat models that contain point mutation(s), small reporter insertions and conditional knockout.
We can generate rat models with several gene expression modalities with high success rate. Some of the models in our portfolio includes:
- Constitutive knock-in, knockout models; point mutation rat models
- Conditional knockout rat models (Example: LoxP- Cre)
- Inducible expression rats (Example: Tet-regulatory systems)
- Humanized rat models (large gene insertion)
- Transgene overexpression models
- Reporter gene insertion
- Cre-driver rats: we can generate Cre-rats using ANY promoter-of-choice (if sequence available)
Timeline for CRISPR Knock-Out or Knock-In Rat Generation
|1. Targeting DNA Vector Creation||A report on cloning and validation|
|gRNA Design and Construction (2-4 gRNAs)||3-4 weeks|
|gRNA In Vitro Functional Validation (2-4 gRNAs)||2-3 weeks|
|Donor DNA Construction||2-6 weeks|
|in vitro Transcription and QC for Microinjection||1 week|
|2. CRISPR DNA Pronuclear Microinjection||1-2 months||A report on microinjection and embryo implantation|
|3. Animal Care, Housing, and Genotyping||3-5 months||At least 1 founder|
|Pups generated and genotyping showing the proof of precise gene insertion||A final report on the project including the original targeting strategy and microinjection details|
|4. F0(s) Breeding to F1(s)
|Includes material purchasing, breeding, housing, and genotyping|
CRE-LoxP Conditional Rat Models for Tissue Specific Gene Knockout Using CRISPR/Cas9 and TARGATT™
Applied StemCell’s employs two complementary technologies to engineer conditional knockout Cre-LoxP rat breeding pairs in a two-step process: the proprietary TARGATT™ and licensed CRISPR/Cas9 gene editing platforms.
- Using CRISPR/Cas9, the gene of interest can be floxed by knocking in LoxP sequences to flank the gene and to generate a conditional knockout rat model (Figure 1; Technical Details).
- The TARGATT™ technology allows any gene of interest (up to 22 kb) to be inserted into preselected and engineered docking sites in the safe harbor locus (H11 locus) of the rat genome. In this case, the Cre recombinase gene can be paired with a promoter of choice (tissue/ cell specific) and inserted into the safe harbor locus for guaranteed expression of the Cre gene driven by the chosen promoter (Figure 2; Technical Details).
When the Cre-rats are thus bred with conditional knockout rats, it results in rat progeny with deletion of floxed gene in the specified tissue (Figure 3; Technical Details).
1. Conditional Knockout Rat Models Using CRISPR/Cas9
Conditional knockout (CKO) animal models are gaining popularity as they circumvent the impediments of constitutive knockout models such as embryonic lethality, compensatory mechanisms and undesired phenotypes and model human diseases better. The most commonly used CKO system is the Cre-LoxP system, where the gene of interest (targeted exons) is flanked by two LoxP sequences (also called floxed allele). The flanking LoxP sequences are inserted at specific sites on either side of the gene of interest using CRISPR/Cas9 technology (Figure 1). The LoxP sites are a target for the Cre Recombinase which catalyzes the deletion of the floxed exon(s).
Figure 1. The schematic describes the first stage in developing a conditional knockout rat model CRISPR to generate a floxed (loxP flanked exon) rat. A single stranded donor DNA (ssDNA) is used for delivering the floxed targeting exons to replace the wildtype form. The donor contains two LoxP sequences flanking the targeted exon(s) along with 5' and 3' homologous arms for directing a site-specific homology directed repair. The donor ssDNA is delivered along with Cas9 (mRNA or protein) and validated gRNAs via microinjection.
2. Cre-driver Transgenic Rat Models Engineered Using TARGATT™ Technology
Cre- rat models are generated by microinjection of an integration cocktail into the pronuclus of TARGATT™ rats engineered with "attP" docking sites at a preselected locus. The integration cocktail consists of the targeting vector (promoter+ Cre gene + attB sequence) and in vitro transcribed PhiC31 mRNA. The integrase catalyzes the recombination between the attB and attP sites, resulting in integration of the promoter-Cre transgene in a site-specific manner without any position effects associated with random insertion. The attB-attP recombination results in unique sequence (attL and attR) flanking the inserted transgene which is not recognized again by the integrase and thereby ensures an uni-directional, stable integration reaction.
Figure 2. Schematic illustrates the engineering of a Cre-driver rat model using TARGATT™ integrase technology. A cocktail of TARGATT™ donor vector carrying the integrase recognition sequence “attB” (orange arrow) and the Cre-driver transgene (promoter-of-choice; yellow triangle and Cre gene; dark blue), and the TARGATT™ integrase is microinjected into the pronucleus of a TARGATT™ rat embryo that carries an “attP” docking site (purple arrow) inserted into a preselected safe harbor locus such as H11 (described earlier). The Integrase catalyzes a recombination between the attP and attB sites, resulting in two new hybrid sites, attL and attR which are no longer recognized by the integrase enzyme. As a result, gene integration is stable and the process is highly efficient in generating transgenic Cre rats.
Conditional knockout rats are generated by crossbreeding the two transgenic rat lines: (a) the homozygous “floxed” (flanked by loxP) allele rat model, and (b) the Cre-driver rat model with tissue specific expression or ubiquitous expression (Figure 3). The Cre expression has minimal unwanted effects in the animal as the mouse genome does not contain endogenous loxP sites, providing an ideal background for site-specific recombination.
CRISPR-Cas9 technology will be used to generate rat models that contain point mutation(s), small reporter/ gene insertions and conditional knockouts. 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.
Case Study #1: Rat knockout using CRISPR/Cas9
Case Study #3: Rat knockout using CRISPR/Cas9
Goal: To generate a knockout rat model using CRISPR/Cas9 in Sprague Dawley (SD) strain of rats.
To achieve this model, a cocktail of gRNA and Cas9 mRNA was injected into the cytoplasm of SD rat embryos and implanted in SD foster rats. Pups born after microinjection were screened for deletion genotypes using PCR and 8 animals which showed PCR fragments smaller than that of a wild type (wt) were chosen for sequence analysis. Out of 33 rats born, six rats were confirmed as founders with deletion in exon 2 of the gene of interest.
Figure 3. PCR amplification and sequence analysis of a CRISPR- based rat knockout model. (a) PCR amplification of the exon 2 region of the gene of interest initially identified 8 rat pups as possibly containing deletion mutations (#1, 3, 5, 15, 18, 24, 26, and 30; denoted by *) based on the smaller PCR fragments than that of a wild type (wt). (b) The predominantly lower bands of the PCR products for the potential founders were excised and submitted for Sanger sequencing. A representative deletion pattern based on sequencing data from rat #30 is shown. The deletion mutation for this rat spans intron 1 to exon 2, likely affecting the splicing machinery which in turn could severely damage mRNA formation and translation.
- 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, 309–312
CRISPR Knockin H11 Locus in Pigs:
- Ruan, J., et al. (2015). Scientific reports, 5:14253. doi: 10.1038/srep14253