CRISPR/EGE™-based Gene Editing

CRISPR (clustered, regularly interspaced, short palindromic repeats) is a defense mechanism in bacteria that degrades invasive viral DNA and other exogenous DNA. The CRISPR/Cas9 system from Streptococcus pyogenes is currently the most commonly used one.

The Cas9 protein first binds to the CRISPR RNA (crRNA) and TRACER RNA (tracrRNA) to form a complex, and then binds to the target sequence to form the RNA-DNA complex, which ultimately makes double strand breaks in the DNA.

crRNA recognizes target DNA sequences and tracrRNA is a conserved essential component for Cas9 activity. In order to simplify gene-editing procedures in the laboratory, crRNA and tracrRNA are fused together to make Single Guide RNAs (sgRNAs). The target gene can be edited by transfecting cells with a plasmid that expresses both the sgRNA and the Cas9 protein. The CRISPR/Cas9 technology has been successfully used in bacteria, yeast, plant, fish and mammals, and is the most efficient genome-editing technique.

The homologous recombination (HR) efficiency between exogenous DNA and a target genome is very low using the standard CRISPR/ Cas9 technology. To improve HR efficiency, BIOCYTOGEN developed an Extreme Genome Editing (EGE™) System which increases the HR rate 10-20 fold over standard CRISPR/Cas9 technology. This makes gene editing with the EGE™ system fast and convenient. EGE™ can accurately edit DNA sequences at almost any genomic locus, and is ideal for the generation of a variety of mouse genetic models.

Advantages:

High Speed The F0 generation positive mouse can be obtained in two months at the earliest, while the F1 generation can be obtained in five months
Zero Risk Supported by a strong production platform, customers will be fully refunded if the project fails
High Efficiency Compared with standard CRISPR/Cas9 technology, the efficiency of homologous recombination mediated by the EGE™ system is increased by 10-20 fold
High Quality Soutern blot screening minimizes follow-up experimental risks caused by random insertions
Diversification Conventional knockout, conditional knockout, and gene knockin, etc.
No Species Limitation Cell line, mouse, rat, pig, monkey, and zebra fish, etc.

 

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Southern blot to screen out random insertions

Statistical analysis shows that random insertions (from the targeting vector) occur in 32% of CRISPR/Cas9-based EGE™ projects. Of these animal models containing insertions, 14% cannot be removed even by breeding.  Southern blot analysis is the gold standard to test for random insertions. Biocytogen recommends Southern blot analysis to ensure that there are no random insertions in our gene edited animal models.

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