Journal
NATURE CELL BIOLOGY
Volume 20, Issue 11, Pages 1315-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41556-018-0202-4
Keywords
-
Categories
Funding
- Genome Tagging Project
- Chinese Academy of Sciences [XDB19010204, OYZDJ-SSW-SMC023]
- National Natural Science Foundation of China [31530048, 81672117, 31730062]
- Shanghai Municipal Commission for Science and Technology [16JC1420500, 17JC1400900, 17JC1420102, 17411954900]
Ask authors/readers for more resources
CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein-protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available