China builds first zebrafish directional mutant library 1/4 related to human disease

Nearly 20 years ago, scientists began sequencing the zebrafish genome, a favorite pattern of patterned organisms by biologists working on the early development of the brain and other organs. As its genome was declassified, zebrafish became the animal of choice for studying human diseases and exploring disease treatment.

China builds first zebrafish directional mutant library 1/4 related to human disease

Recently, the results of the “Zebrafish Chromosome 1 Full Gene Knockout Program” by Chinese scientists were published online in the international journal Genome Research. This is the first large-scale construction and genetic screening program of the large-scale zebrafish reverse genetic mutant library.

The authors of the paper are from 24 institutions, including the Institute of Aquatic Biology of the Chinese Academy of Sciences, Tsinghua University, the Institute of Zoology of the Chinese Academy of Sciences, and Peking University, the Zebrafish Chromosome 1 All-Gene Knockout Alliance (ZAKOC). The first author of the paper is the Institute of Aquatic of the Chinese Academy of Sciences, the first author is Sun Yonghua, a researcher of the Institute of Aquatic sciences of the Chinese Academy of Sciences, the communication author is Liu Feng, a researcher of the Institute of Animals, a member of the Chinese Academy of Sciences, Professor Meng Anming of Tsinghua University, and Zhu Zuoyan, a researcher of the Institute of Aquatic sciences of the Chinese Academy of Sciences.

Zebrafish is an important model animal to carry out research in life science, health science, environmental science and so on. The zebrafish genome is about 1.5Gb in size and contains 25 chromosomes and about 32,000 genes. Of these, chromosome 1 is about 60Mb in size, accounting for 4% of the zebrafish’s total genome.

In the history of zebrafish research, European and American scholars have launched several large-scale random mutagenic mutants. In February 2013, a new generation of gene editing technology CRISPR/Cas9 system was introduced, the Chinese zebrafish community, that is, the spontaneous organization of the Zebrafish Chromosome 1 whole gene knockout program (ZAKOC), the use of this technology to carry out large-scale, full-chromosome level systematic gene knockout of the zebrafish genome, the world’s first start using the technology implementation model animal whole genome knockout research program.

Using CIRISR/Cas9 technology, the ZAKOC Alliance systematically knocked out 1,333 genes on the zebrafish chromosome 1, successfully knocking out 1,029 of them and obtaining 1,039 passable variants for 636 genes.

This research, which lasted more than six years, for the first time realized the systematic gene knockout of the entire chromosomes of vertebrates, not only led the international zebrafish phenotype group research, but also gave birth to China’s first large-scale zebrafish directional mutant bank.

Currently, all mutant lineage and genetic information is made available to academia through the National Aquatic Species Resource Library, the National Zebrafish Resource Centre (http://www.zfish.cn/TargetList.aspx).

The program produces the largest experimentally proven library of effective and ineffective gRNA sequences in academia, and through big data analysis, it reveals a close positive correlation between the knock-out efficiency of gRNA and its target GC content. Of all mutants, about a quarter are associated with human disease.

The paper also shows a number of representative mutants and their phenotypes, and finds that pure-fit mutants are inconsistent with gene-knocking phenotypes, further enriching the recently discovered theory of “genetic compensation effects”.

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