A method of efficient regulation of endogenous mRNA translation through CRISPR/Cas9.

Genome editing is an efficient biotechnology method for accurate and directional modification of genes at the genome level. the emergence of
simple and efficient CRISPR/Cas9 editing system has brought about a new technological revolution in life science.
CRISPR/Cas9 usually results in the addition or deletion of DNA bases at genomic target sites, resulting in the loss of gene function.
recently, the Gao Caixia Research Group of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences established a method for efficientregulation of endogenous mRNA translation through CRISPR/Cas9.
this method can increase the level of the encoded protein of the target gene by improving the efficiency of protein translation. The expression products of
protein-coding genes are generally regulated by transcribed, post-transcriptal RNA processing, protein translation and post-translation processing, protein degradation and so on.
mRNAs for eukaryotic cells consist of 5' non-translated regions (5'Untranslated Region, 5'UTR), Open Reading Fragments of the encoded protein (Open Reading Fragment) and 3'Untranslated Regions (3'Untranslated Region, 3'UTR).
study found that 5'UTR has open reading boxes with translation capabilities, called upstream Open Reading Box (upstream Open Reading, uORF). corresponding to
, the open reading box after 5'UTR is called the Primary Open Reading Fragment, pORF).
uORF is usually able to inhibit downstream pORF translation.
bioinformatics analysis shows that uORF is widely present in plants and animals, and more than 30% of mRNAs in humans, mice, plysing mustard, rice and corn contain predicted uORF, but there is also a lack of efficient and sophisticated methods for functional research and genetic manipulation of uORF.
High Caixia research team used CRISPR/Cas9 to edit uORF and found that it significantly improved the translation efficiency of the target gene.
, using CRISPR/Cas9 to edit the uORF translation of the starting region and surrounding sequences of the four genes in the amoeba and lettuce, obtained a number of corresponding gene uorf mutants.
the mRNA translation levels of the target genes of these uorf mutants have improved to varying degrees.
, uORF, upstream of the key gene GGP (GDP-L-galactose phosphorylase), which is a mutation in vitamin C synthesis, can increase vitamin C content in lettuce leaves by about 150%.
use crispR/Cas9 editing uORF translation starting area will appear two results: (1) completely destroy the translation starting ability of uORF resulting in the loss of uORF function;
this study shows the application prospects of manipulating protein levels in plant molecular biology research and genetic breeding through genome editing uORF manipulation of mRNA translation. In addition,
, the approach may become more widespread and easier to operate as new genomic editing tools emerge and methods are further optimized.
because uORF is common in animal and plant genes, this method also has a broad application prospect.
results were published in Nature-Biotechnology on August 6.
Gao Caixia Research Group Associate Researcher Zhang Huawei, Ph.D. graduate student S. Xiaomin, Ji Xiang as the paper co-first author.
the research was supported by the Ministry of Science and Technology, the National Natural Science Foundation Foundation Basic Science Center and the Chinese Academy of Sciences.
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