C-CRISPR" technology: One-step access to genes completely knocked out of mice and crab-eating monkeys.

On June 6th Cell Research published a study online that used improved "C-CRISPR" technology to get mice and monkeys completely knocked out of single or multigene function.
The study was carried out by a team of Yang Hui of the Institute of Neuroscience of the Shanghai Institute of Life Sciences of the Chinese Academy of Sciences, Yang Hui of the Center for Excellence and Innovation in Brain Science and Intelligent Technology, Xiong Zhiqi Research Group, and Sun Qiang, a non-human primate research platform in Suzhou, Neurological Institute.
The study, by injecting multiple continuous guided RNA (sgRNAs) for exogenous exterminations into fertilized eggs, was able to make high-volume genes that completely knocked out mice and were used for dedict analysis, and quickly produced genetic knockout monkey models.
CRISPR/Cas9 system is a very efficient method of gene editing, but most of the animals that have been genetically edited have chitility, meaning that only a portion of their cells' genes have been edited.
for studies involving dnotypes, chimed-in gene-edited animals need further hybrid breeding to get a complete genetic knockout of the animal.
When it comes to large animals, such as non-human primates, the problem of chimed-ins is particularly acute because macaques breed for 5-6 years and have only one cub per birth.
many researchers have tried a one-step process to produce genetically modified animals that are not embedded, especially large animals.
include injecting Cas9 mRNA and sgRNA into ovaries instead of fertilized eggs, or injecting Cas9 proteins, or injecting double sgRNA.
but DNA sequencing analyzed the animal tissues of these methods and found that they were quite inefficient in fully removing the double allergens.
further to get the genes completely knocked out of the mice and crab-eating monkeys.
map of the targeting bits of the Tyr gene sgRNA in mice.
B. injection of Cas9 mRNA combines multiple adjacent sgRNAs into the fertilized eggs of mice.
map of the targeting point of the C. Crab Monkey Prrt2 gene sgRNA.
D.MII egg single sperm injection develops into the primary nuclear fertilized egg, followed by a Cas9 mRNA injection to combine multiple adjacent sgRNAs.
in this study, the researchers found that a cocktail-style mixture of Cas9 mRNA and multiple sgRNAs injected into fertilized eggs allowed 100% complete deletion of single or multiple genes in mouse embryos and 91%-100% deletion in monkey embryos.
method can produce both insertion deletion and extation deletion, thus achieving efficient gene complete knockout.
researchers also used the C-CRISPR method to establish F0-generation mice with complete knock-out of genetic function, for rapid dedgy analysis of gene function, and for F0-generation monkeys with complete knockout of gene function.
, a high-pass sequencing analysis showed that C-CRISPR would not introduce significant off-target changes in genetically edited mice and monkeys.
The work was carried out by postdoctoral fellow Zuo Yiwei, assistant researcher Cai Yijun, postdoctoral researcher Li Kui, graduate student Wei Yu, postdoctoral wang Bang'an in the primate disease model research group researcher Yang Hui, disease neurobiology research group researcher Xiong Zhiqi and Under the guidance of Sun Qiang, director of Suzhou Non-Human Primate Research Platform, the other members of the research group actively participated, and received the strong assistance of Li Jinsong and Xu Guoliang, researchers of the Institute of Biochemistry and Cell Biology of Shanghai Academy of Health Sciences.
this work has been approved by the Chinese Academy of Sciences Strategic Pilot Science and Technology Special (XDB02050007, XDA01010409), the National High-Tech Research and Development Project (863 projects; 2015AA020307), the National Natural Science Foundation of China (NDSS) 3152037 and 31500825), and the China Youth 1,000 People Program (Yang Hui) Major Breakthrough Projects of the Chinese Academy of Sciences, Shanghai Youth Science Talent Training Program (15YF1414700), National Key Technology Research and Development Projects (2014BAI03B00, Sun Qiang), Shanghai Science and Technology Commission Project (16JC1420202, Yang Hui; 14140900100, Sun Qiang), Chinese Academy of Sciences 100 People Program (Sun Qiang) and other projects.
.