CRISPR-Cas9技术高效 z-bo 2020-11-13 415

　　In order to use the CRISPR-Cas9 gene editing system to cut genes, one must design an RNA sequence that matches the DNA of the target gene. Most genes have hundreds of such sequences that differ in activity and uniqueness in the genome. Therefore, finding the best sequence is difficult to achieve manually. A new "CrispRGold" program helps scientists identify the most efficient and specific RNA sequences. This procedure was developed by a research team led by Professor Klaus Rajewsky, a scientist at the Max-Delbrück Center for Molecular Medicine in Germany. The team also developed a new model mouse that already carries the Cas9 protein. Combining this model mouse with these reliable RNA sequences resulted in efficient gene inactivation in primary cells. This allows researchers to discover new genes involved in immune cell regulation.

　　For many molecular biologists, the discovery of this CRISPR-Cas9 gene editing system represents a new milestone in research: Finally, genomic DNA can be cut with high efficiency and precision, which can inactivate genes, Retouch or re-import.

　　This only requires an RNA fragment that brings the Cas9 enzyme to the DNA site to be cleaved. This RNA fragment is called a single guide RNA (sgRNA) and contains a 20-base (A, U, C, or G) sequence complementary to the target site of the genome. Before that, scientists had to select sgRNA manually or laboriously or using a variety of online tools. In some cases, people are not sure whether the selected sgRNA carries the Cas9 enzyme to the appropriate site on the genome or a similar but undesirable site, and whether the efficiency of this sgRNA is relatively high.

　　This new "CrispRGold" program makes it easier to disable specific genes. It looks for a certain DNA target sequence to identify the best site for cleavage and suggests a unique sgRNA sequence that only delivers Cas9 protein to the desired site in genetic material and then can cleave the target gene so that it cannot. Function. This program was developed based on experimental data and the uniqueness and other properties of these sequences.

　　In cooperation with Dr. Van Trung Chu, Robin Graf, a PhD student from the team led by Professor Rajewsky, tested this system in mouse B cells. These cells cannot be cultured in vitro for a very long time because they will not survive for a long time outside of their natural environment. Therefore, genes must be inactivated as quickly as possible in many B cells in order to study their functions. Chu does this by cultivating a genetically modified mouse strain that produces large but well-tolerated Cas9 proteins.

　　The researchers then isolated B cells from these mice and transported individual gene-specific sgRNAs to these cells. Graf said that the sgRNA designed using the CrispRGold program destroys these target genes with a high repetition rate in an average of 80% of these cells. "In this type of low-throughput experiment, high efficiency and low error rate are absolutely necessary."

　　The researchers used their new method to identify many previously unknown genes involved in the development of B cells. Graf said that the CrispRGold program will now be posted online so that it can be used by scientists all over the world: "This program can easily be used in other types of cells from a range of organisms. It may also be used in It is of great significance in clinical application-it regards sequence uniqueness as a high priority factor, thus minimizing the modification of potentially unwanted genes, which must be avoided at all costs in gene therapy."