动物实验,CRISPR z-bo 2020-09-28 572

　　Recently, researchers from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences and Shanghai University of Science and Technology have developed an intron-based genome editing method that uses the CRISPR/Cas9 system to achieve efficient zebrafish gene knockout.

　　Du Jiulin is a researcher at the Institute of Neuroscience, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. He graduated from the University of Science and Technology of China in 1993, and received a doctorate from the Shanghai Institute of Physiology, Chinese Academy of Sciences in 1998. He is a member of the 100 Talents Program of the Chinese Academy of Sciences. His main research directions are sensory integration and learning and memory behavior molecules, synapses, neural circuit mechanisms and blood vessels. Neuroregulation of development and function.

　　Knock animals are a versatile tool for biological research. For example, researchers often use knock-in animals to understand the role of lethal genes in later embryonic function. That is, loxP is inserted into the target genome site to create conditional knockout animals. The specific cell fluorescent protein labeling or endogenous protein via knock-in provides a powerful way to track the dynamics of these cells or proteins in vivo. Zebrafish is a new vertebrate model in life science research. Studies on zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN) or zebrafish's functional genome editing through the CRISPR/Cas9 system have been carried out, but the gene knock-in method is still in its infancy. At present, There is still a lack of feasible methods. Inserting large DNA sequences into specific genomic sites has become a bottleneck in zebrafish related research.

　　In April this year, the research team reported a new gene knock-in method in "Cell Research". In this new study, they developed CRISPR/ by using mouse gene knock-in via homology-induced repair (HDR) and cell culture donor gene integration via NHEJ (homologous end binding). The effective zebrafish gene knock-in strategy through Cas9 can be widely used to label different cell types and label endogenous proteins. The researchers used this strategy to specifically label dopaminergic neurons, serotonin-activated neurons, glial nerve cells and endothelial cells. The researchers also successfully added the EGFP tag to the carboxyl end of endogenous glial fibrillary acidic protein. In this gene knock-in system, the researchers selected an sgRNA target from the introns of the target gene, added the DNA sequence of the sgRNA target site to the 3'intergene region of the target gene, and added it to the donor In the plasmid. As a homology arm. This strategy maintains the complete reading frame and 5'and 3'regulatory elements of the targeted endogenous gene, thereby maintaining the integrity of the target gene.

　　In addition, the intron design of the sgRNA target avoids incorrect integration into exons caused by NHEJ. Compare this method with the exon-based method because all insertion events are within the frame. It can increase the chain efficiency by 3 times. It is very important to add the right arm (ie the 3'intermediate gene region of the target gene) to the donor plasmid. According to this study, the length of the plasmid arm is not important for the preparation of the donor, as long as the arm sequence meets certain requirements: 1) sgRNA must be contained in the intron part of the left arm; 2) contains all mRNA 3'UTR The 3'intergenic region of the sequence must be contained in the right arm. Another important factor for successful gene knock-in is the efficiency of sgRNA cleavage. Several sgRNAs can be designed to target the intron sequence of the left arm. For knock-in experiments, it is necessary to select the sgRNA with the highest cutting efficiency.