公开(公告)号：US20230175019A1

公开(公告)日：2023-06-08

申请号：US17927454

申请日：2021-05-28

Applicant: University of Southern California

Inventor： Scott E. Fraser ,  Le Trinh

IPC: C12N15/90 ,  A01K67/027 ,  C12N15/113 ,  C12N15/65 ,  C12N15/79

CPC classification number: C12N15/902 ,  A01K67/0275 ,  C12N15/113 ,  C12N15/65 ,  C12N15/79 ,  A01K2217/072 ,  A01K2227/40 ,  A01K2267/03 ,  C12N2310/20 ,  C12N9/22

Abstract: A new DNA knock-in approach is provided based on the usage of three single guide RNA (sgRNA) to increase the integration efficiency of donor DNA based on the CRISRP-Cas system. The approach uses a pair of universal sgRNAs complementary to the donor DNA and a single sgRNA that targets the locus of interest. In various embodiments, targeting is achieved by pre-forming a DNA:RNA:protein (DNA:RNP) complex in vitro and introducing the complex into the embryo or cells of interest either by microinjection or transfection.