1. Brief introduction of EGETM technology
The CRISPR (clustered, regularly interspaced, short palindromic repeats) is an immunologic mechanism from bacteria that degrades invasive virus DNA or other exogenous DNA. In bacteria and archaebacteria, the CRISPR system is divided into three types, where the type I and type III systems needs multiple CRISPR-related proteins (Cas protein) to play a role, while the type II only needs the Cas protein, which provides wide application convenience. The CRISPR/Cas9 system from Streptococcus pyogenes is currently the most commonly applied one.
There are two nuclease domains in the Cas9 protein that can cut the two DNA single strands. The Cas9 protein binds to the crRNA and tracrRNA to first form the compound, and then invades the DNA by binding to PAM sequences and forming the RNA-DNA composite structure to cut and break the targeted DNA double strands.
The PAM sequence is widely used for its simple structure, and plenty of target sequences can be found in almost every gene. The CRISPR/Cas9 technology is successfully used in the cells of plant, germ, yeast, fish and mammals, and is the most efficient genome-editing technique.
Researchers genetically engineer the crRNA and tracrRNA, and connect them to obtain the sgRNA (single guide RNA). The fused RNA has activity similar to the wildtype RNA, and is convenient due to its simplified structure. The plasmids expressing both sgRNA and Cas9 can be obtained by placing the components expressing sgRNA and Cas9 into the same vector. The target gene can be affected after transfecting cells with the plasmids.
The homologous recombination efficiency of exogenous DNA is very low when using the current CRISPR/Cas9 technology for gene knockin. To improve the recombination efficiency, BIOCYTOGENTM developed and optimized an efficient gene knockout/knockin system. Biocytogen Extreme Genome Editing System (EGETM) is based on the CRISPR/Cas9 technology. The efficiency of homologous recombination mediated by the EGE system is improved 10 to 20 times in comparison to general CRISPR/Cas9 technology, which makes gene engineering with the EGETM system faster and more convenient. The EGETM system can edit the DNA sequence accurately at almost every genomic locus, which can achieve conditional knockouts, whole-gene knockouts, gene knockins, and gene mutations. BIOCYTOGEN registered the “EGETM” brand successfully in September 2015.
2. EGETM system preparation model animal process figure
3. Service process
BIOCYTOGENTM provides services to construct a mouse model in one step, including model design, construction, and genotypic identification service.
3.1 Project feasibility evaluation and model design
BIOCYTOGENTM possesses a senior technical team specializing in comprehensive analysis of project feasibility, and provides a free and professional model design service. The services include:
Ÿ Define the purpose for constructing the model
Ÿ Analyze the target gene’s structure and sequence information
Ÿ Analyze existing data
Ÿ Design all possible model construction schemes
Ÿ Analyze the advantages and risks of all model construction schemes
Ÿ Accurate prediction of project implementation time and quotation
3.2 Designing and construction of targeting vector
Ÿ Design the project and primer synthesis
Ÿ Construction of CRISPR/Cas9 plasmid
Ÿ sgRNA activity detection
Ÿ Targeting vector construction
Ÿ CRISPR/Cas9 transcription in vitro to prepare mRNA
3.3 Pronuclear injection
Ÿ Inject the CRISPR/Cas9 mRNA into the mouse zygotes
3.4 Obtain the mouse of generation F0 and breeding
Ÿ Recipient female mouse feeding and the birth of chimeric mouse
Ÿ Mating of chimeric mouse and wild mouse
3.5 Obtain the phyla-genetic hybrid mouse of generation F1
Ÿ Birth of generation F1 mice
Ÿ Ensure the phyla heredity by identification of generation F1 mouse tail genotype
3. Advantages of the EGETM system
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 strong production platform, and customers will be fully refunded if the project fails
High efficiency: compared with current CRISPR/Cas9 technology, the efficiency of homologous recombination mediated by the system is improved 20 times
High quality: Southern blots ensure quality, and minimize follow-up experimental risks caused by random insertion
Diversification: conventional knockout, conditional knockout, and gene knockin, etc.
No species limitation: cell line, mouse, rat, pig, monkey, and zebrafish, etc.
4.1. Gene knockin rat
BIOCYTOGEN developed the CD4-dsRed and FoxP3-DTR-EGFP reporter gene knockin rat using the EGE system in February 2014. Since then, BIOCYTOGEN has made 70 kinds of gene knockout rat, conditional knockout rat, and gene knockin rat models, which means BIOCYTOGEN is on the leading edge of the technology development and invention of gene knockout/knockin rat models in both the domestic and international markets. BIOCYTOGEN has prepared more than 200 kinds of gene knockout/knockin rat/mouse models for customers using the EGE system.
4.2 Gene knockin cell line
BIOCYTOGENTM used the U2OS cell line to knock in an EGFP (fusion protein) at the translation initiation site of the ACTB (cytoskeleton filiform protein) gene coding frame. We also used the rat C6 cell line to knock in an EGFP (fusion protein) at the translation initiation site of the LMNB1 (nuclear envelope protein) gene coding frame.
After assessing recombination efficiency using flow cytometry, the efficiency of EGFP-ACTB gene knockin mediated by CRISPR/Cas9 is only 1.91% in U2OS cell lines, while the EGE knockin efficiency can reach 15.02%, which shows 8 times improvement. In the C6 cell line, the EGE system improves the knockin efficiency of EGFP-LMNB1 to 3.6% from 0.19%, which shows 19 times improvement.