Artificial sperm semi-cloning technology: important gene Dnd1 to achieve the individual horizontal amino acid function site of genetic screening.

On October 1st, the international academic journal Nature Cell Biology published online the latest collaborative study by Li Jinsong and Chen Yong of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences, "CRISPR-Cas9-mediated base-editing screening in mice-research-research-dND1 amino acids that are all-a-reison for the primordial germ cell."
the work uses the latest CRISPR/Cas9-mediated monobase editing system (BE3) in combination with semi-cloning technology mediated by haploid embryonic stem cells ("artificial sperm") to achieve genetic screening of the important gene Dnd1 that affects the development of primitive reproductive cells (PGCs) in mice.
CRISPR/Cas9 gene editing system uses Cas9 nucleic nucleic endoenzymes to bind sgRNA to cut the target DNA, inducing DNA to perform non-homologous end connection (Non-homologous End Joining, NHEJ) or homologous recombination (Homology-directed repair, HDR) damage repair mechanism, and thus to achieve target gene sequence mutation.
2016, David R. Liu's team established the highly efficient lysacin monobase editor (BE3) based on CRISPR/Cas9 technology to induce single nucleotide mutations, which can target the target point C? G to T? A (Komor et al., 2016, Nature).
because the system avoids the damage that traditional CRISPR/Cas9 can cause from double-stranded DNA fractures, it was quickly used by scientists at home and abroad to edit genes in different species. In addition to the gene editing of a single amino acid site, the single-base editing system in
can theoretically carry out the study of protein structure and function through the genetic screening of amino acid sites, but this application has not been reported.
Li Jinsong's research group has been engaged in the study of mouse monoploid embryonic stem cells and their applications.
2012, Li Jinsong's research team, in collaboration with Xu Guoliang's research team, established the first lone male monoploid embryonic stem cells, and demonstrated that these cells can replace sperm to "fertilize" oocytes to produce semi-cloned mice, although the technology produces semi-cloned mice (Yang et al., 2012, Cell).
2015, Li Jinsong's research team and Yang Li's research team collaborated to obtain lonemale monoploid embryonic stem cells ("Artificial Sperm", 2015, cell cell) by removing two imprinted regulatory regions (Differentially Methylated Region, DMR) H19-DMR and IG-DMR.
they speculated that the "artificial sperm"-mediated semi-clone technique combined with BE3 technology had the potential to achieve in-body genetic screening of specific protein-critical amino acids.
, the researchers first established an efficient single-base editing system in the embryonic stem cell line of mice, by adding an approved bit sequence to the N and C ends of the base editor (BE3). After
, they applied the system to mouse "artificial sperm" and found that optimized BE3 not only enabled efficient single-base editing on "artificial sperm", but also injected BE3-carrying "artificial sperm" into the egg and also efficiently produced pure dot mutations in semi-cloned mice.
they then imported a sgRNA slow virus library (containing 77 sgRNAs) into the "artificial sperm" and found that they could efficiently induce single-base mutations at different sites in semi-cloned mice.
two rounds of genetic screening, they found and verified that DND1's E59K, V60M, P76L and G82R play edimen in the development of PGCs.
they then found that these sites play a key role in the stability of DND1 proteins and their interactions with other proteins through multiple hierarchical analyses of protein structure prediction, post-mutation protein stability, protein-protein interactions, and many other levels.
the work for the first time in the world, the use of base editing technology to achieve the genetic screening of the key amino acid function sites of individual level proteins, for the study of protein structure and function opened up a new system. another potentially important application of the
system is to screen functional sites of human disease-related genes and compare them with existing SNVs databases to predict the site of disease-related genes.
the work was completed by Li Qing, a doctoral student of Li Jinsong group, and Li Yanxuan, a postdoctoral fellow in Chen Yong group, under the joint guidance of researchers Li Jinsong and Chen Yong.
the work has been supported by the Ministry of Science and Technology, the State Fund Committee, the Chinese Academy of Sciences Class B pilot special project and the Shanghai Science and Technology Commission funding, and the Center for The GtP (Whole Genome Labeling Program) and animal experimental technology platform of the Institute of Biochemistry and Cell.
Source: Shanghai Institute of Life Sciences.