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CRISPR-Cas9 and Mouse Models

CRISPR/Cas9 genome editing technology has revolutionized the way to create animal disease models. Scientists have used this technique numerous times to study the effects of certain genes on the mouse genome. And knockout mice provide impressive insights into the internal workings of the human genome because of the similarity between mouse and human and the effectiveness of CRISPR technology. <br><br>https://www.creative-biogene.com/crispr-cas9/products/knockout-mice-659.html

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CRISPR-Cas9 and Mouse Models

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  1. CRISPR/Cas9 and Mouse Models info@creative-biogene.com https://www.creative-biogene.com/crispr-cas9/

  2. CONCENTS CRISPR/Cas9 Information 01 01 Mouse Models 02 02 CRISPR/Cas9 Mice Applications 03 03

  3. Information CRISPR/Cas9 system A genome editing tool that is creating a buzz in the science world; Adapt from a naturally occurring genome editing system in bacteria; Edit gene by removing, adding or altering sections of the DNA; Allows for the introduction or removal of more than one gene at a time; Enable researchers to edit parts of the genome in an easy way; Not species-specific, can be used in almost cell lines and most animal; Already being explored for a wide number of applications. High Faster Cheaper Versatility

  4. Information | Components Guide RNA Guide RNA Cas9 Cas9 dCas9 dCas9 Cas9 follows the gRNA that directs the Cas9 to the same position in the DNA sequence and cleave the two strands The gRNA is designed to find and bind to a specific sequence in the DNA. dCas9（another type of Cas9 lacking nuclease activity but retaining DNA binding activity) can be used for transcriptional activation/inhibition (CRISPRa/CRISPRi), epigenetic modification and CRISPR/Cas9- meditated ChIP.

  5. Information | CRISPR/Cas Systems

  6. Information | Principles Target Cleavage (DSB) The DSB can be repaired in a NHEJ way or HDR way depending on whether homologous arms exist. HDR NHEJ Knockout Repair template with homology arms Deletion/Insertion  NHEJ: non-homologous end joining  Insertion Point mutation Knockin  HDR: Homology Directed Repair 

  7. Mouse Models Mice have been very useful in the laboratory, and the first choice for studying mammalian biology. Knockout mice A A Knockin mice B B Point mutation mice C C

  8. Mouse Models | Knockout Mice Conventional knockout mice When gRNA(s) designed for specific sites and Cas9 mRNA are simultaneously injected into mouse single-cell embryos, cutting and incomplete repair (by NHEJ) at the target sites may result in gene insertion or deletion. If the cleavage site is located in the coding region of the gene, this may lead to frameshift mutation downstream of the site, resulting in conventional knockout. Conditional knockout mice The ssDNA containing two LoxP sequences located at the 5' and 3' homology arms is simultaneously injected into mouse single-cell embryos with Cas9 mRNA and gRNA. ssDNA acts as a DNA repair template that will lead to the two LoxP sequences insert into the targeted genes (by HDR).Finally, founder mice with Loxp sequences are obtained and mated with mice that specifically express Cre, and their progeny are genotyped to obtain F1 mice with loxP and Cre enzymes that are tissue-specific knockout mice.

  9. Mouse Models | Knockin Mice Target gene loci Knockin Target sequence (<200bp), DNA oligonucleotides are suggested to be donor DNA. This approach is generally used to insert marker genes, stop regions for specific functional regions, and loxP sequences in conditional insertion models. Larger DNA sequence (>200bp), a donor vector is more efficient. This approach will allow you to insert luminescent or fluorescent reportergenes at specific target locations. Rosa26 Knockin The Rosa26 locus on mouse chromosome 6 is considered to be one of the ideal locations for knockin targeted genes (always for functionalgenes). With the application of CRISPR/Cas9-mediated knockin technology, large DNA fragments can be accurately and efficiently inserted into the Rosa26. the gRNA targets the sequence of the Rosa26 locus, and the donor vector is needed.

  10. Mouse Models | Point Mutation n Point mutation mice are widely used to study the role of specific nucleotides or amino acids in genetic elements or proteins, as well as to mimic human genetic diseases n ssDNA ssDNA is designed to carry the desired mutation n Micro-injection The gRNA, Cas9 mRNA and ssDNA are injected into mouse single-cell embryos simultaneously. n Homology Directed Repair ssDNA acts as a DNA repair template that will lead to one or more nucleotides mutated at the target site during homologous directed repair (HDR)

  11. CRISPR/Cas9 Mice Applications Study the role of transcriptional regulatory elements CRISPR/Cas9 mice Study the functions of specific genes/proteins Verify Cell-type-specific targets drug Drug screening and development Preclinical safety and toxicity studies

  12. Thank You Tel：1-631-626-9181 Email：info@creative-biogene.com

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