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求助，基因敲除工具CRISPR/Cas9和cre-loxp system
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CRISPR/Cas9 作为一种基因敲除工具来说，比现有的基因敲除工具cre-loxp system，它的优越性在哪儿，有没有大神可以告诉我一下，感谢！感谢！
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制作原理CRISPR/Cas9（Clustered Regularly Interspaced Short Palindromic
Repeats）是最新出现的一种由RNA指导Cas核酸酶对靶向基因进行特定DNA修饰的技术。 CRISPR
是细菌和古细菌为应对病毒和质粒不断攻击而演化来的获得性免疫防御机制。在这一系统中，crRNA（CRISPR-derived
RNA）通过碱基配对与tracrRNA（trans-activating
RNA）结合形成双链RNA，此tracrRNA/crRNA二元复合体指导Cas9蛋白在crRNA引导序列靶定位点剪切双链DNA达到对基因组DNA 进行修饰的目的。CRISPR/Cas9技术特点1、可实现对靶基因多个位点或多个基因同时敲除；2、实验周期短，价格低；3、可应用于大小鼠等，无物种限制。
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CRISPR Knock-In, Knock-Out, Conditional Knock-Out Rat
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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 compartmentalizatio (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.
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, point mutation rat models
Conditional knockout rat models (Example: LoxP- Cre)
Inducible expression rats (Example: Tet-regulatory systems)
We can also generate more advanced rat models using Applied StemCell's proprietary :
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
Deliverables
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
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)
&2-5 months
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).&
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- yellow triangle and C 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.&
Figure 3.&Crossbreeding the conditional knock-out rat with a Cre-recombinase expressing rat. The Cre expression is driven by a promoter of choice: tissue specific or ubiquitous promoter. As an example, a CNS-specific promoter is shown in the figure. The expressed Cre recombinase deletes the floxed exon(s) in a spatial specific manner there by causing a frame shift in downstream sequence.&
Case StudiesCase Study #1:&Rat knockout using CRISPR/Cas9
Goal: To generate a knockout rat model by using CRISPR/Cas9 technology to create a deletion within the gene of interest in Sprague Dawley (SD) rat strain.
Sixteen pups were born after microinjection of CRISPR elements targeting the gene of interest, screened for deletions by PCR, and confirmed by sequence analysis. Three pups (#3, 6 and 16) were confirmed to have homozygous deletions and one (#13) was a heterozygous knockout.
Figure1.&Genotype screening for knockout founder by PCR. Sixteen rat pups were tested by PCR using primer sets for the gene of interest and four were confirmed to have the desired deletions (marked by *); the unlabeled lanes contains GeneRuler 1 kb DNA Marker
Case Study #2: Rat Point Mutation Rat model
Goal: To generate a mutant rat model by knocking in an R & H (CGC & CAC) point mutation in a neuronal gene in the dark agouti rat strain.
Two out of 31 rats were positive for the desired point mutation following microinjection of a CRISPR cocktail into rat embryos.
Figure 2. Sequence analyses of point mutation rat model.&(a) Schematic representation of th (b) Representative chromatogram of a founder rat.
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.&
References
CRISPR technology:
Smalley, E. (2016)&
Baker, M. (2014) Gene editing at CRISPR speed.
CRISPR Knockin H11 Locus in Pigs:
Ruan, J., et al. (2015).
Hu, J. K., et al. (2016).&
Besschetnova, TY., et al. (2015). .
McKenzie, CW., et al. (2015).
Bishop, KA., et al. (2016). .
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