Generation Of Genetic Engineering Cas9-Expressing Cell Line And Monkey Model

CRISPR-Cas9 is an emerging gene editing system that can precisely modify targeted sequences.Its emergence has revolutionized the field of biology and promoted current research in medicine,genetics,embryology,bioinformatics,and pathology development.With the of the CRISPR/Cas9 system,we can achieve targeted gene knockout or exogenous base insertion.Compared with ZFN and TALEN gene editing technology,CRISPR/Cas9 system has the advantages of low cost and easy operation,so it has played an important role in plant breeding,animal husbandry improvement and biomedical engineering research.But the size of Cas9 protein caused the low delivery efficiency and further affect editing result on practical application.The Cas9-expressing cell lines and animal model avoid the delivery of Cas9 protein,and simply using the sg RNA can complete gene editing in vivo and vitro,thus simplifying the use step and expanding the vitro application.But so far,most of the Cas9-expressing cell lines and animal models are focusing on mice,and there are only few studies in other model organisms or large animals.The extremely high similarity between non-human primates and humans in genetic background makes them an important material in life science research,and plays an irreplaceable role in disease mechanism research and drug development.Therefore,the efficient application of gene editing technology in non-human primates can greatly promote the development of biomedical research.In this study,we generated a Cas9-expressing macaque fibroblast cell line by lentivirus,and proved that this cell line can easily and efficiently complete targeted sequence editing.And we also tried to generate a Cas9-expressing macaque model to overcome the delivery difficulties associated with Cas9,hoping to provide an attractive model for studying biological processes and diseases.Although the obtained rhesus monkey model had the Cas9 sequence insertion at the genome level,it failed to show the ideal function.We also made an explanation for this situation,hoping to provide guidance and advice for future Cas9 animal model generation work.