TY  - JOUR
A1  - Lu, Hengxing
A1  - Liu, Jun
A1  - Feng, Tao
A1  - Guo, Zihang
A1  - Yin, Yunjun
A1  - Gao, Fei
A1  - Cao, Gengsheng
A1  - Du, Xuguang
A1  - Wu, Sen
T1  - A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor
Y1  - 2021/05/01 
JF  - Genome Research 
JO  - Genome Research 
SP  - 900 
EP  - 909 
DO  - 10.1101/gr.271312.120 
VL  - 31 
IS  - 5 
UR  - http://genome.cshlp.org/content/31/5/900.abstract 
N2  - Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs. 
ER  -