Genome editing allows targeted modifications of the nucleotide sequence of genes, whose advance gains momentum with the development and application of the simple and efficient CRISPR/Cas9 mediated genome editing. At present, research on CRISPR editing is still moving fast, and brings new breakthroughs constantly in many aspects of life science and bioengineering.
Recently, a team led by Professor GAO Caixia at Institute of genetics and Developmental Biology, Chinese Academy of Sciences developed a novel CRISPR editing based approach for fine-tuning the protein translation levels of plant genes, with the potential of being useful in many other eukaryotic organisms.
It is well known that the protein translation levels of eukaryotic genes are generally controlled at multiple layers, e.g., transcriptional regulation, post transcriptional RNA processing, and mRNA stability and translation.
Typically, a eukaryotic mRNA has a 5'-untranslated region followed by the coding region (also called the open reading frame, ORF) and ended with a 3'-untranslated region.
Interestingly, genome-wide bioinformatic analysis discovered the presence of short and upstream ORF (thus named as uORF) in the 5'-untranslated region of diverse eukaryotic genes. It has been estimated that more than 30% of the genes in human, mouse, Arabidopsis, rice and maize may contain one or more uORFs in their 5'-untranslated region.
Several past studies have demonstrated the pivotal importance of a few uORFs in repressing the protein translation of their downstream primary ORFs (pORFs). But the functionality of the vast majority of the uORFs revealed by computation remains unknown. The development of an efficient and facile approach for altering the sequence of uORFs may aid the dissection of these elements, and thus the biological functions of their pORFs.
Inspired by CRISPR technology, GAO's team decided to test if CRISPR/Cas9 mediated editing may be harnessed to rapidly modify uORFs so as to achieve fine tuning of the protein translation levels of important plant genes (Figure).
They introduced a range of nucleotide insertions or deletions into the uORFs of four representative plant genes in Arabidopsis and lettuce via CRISPR editing, and examined their effects on pORF translation and trait performance. As anticipated, the CRISPR mutants of the uORFs showed various levels of increase in pORF translation.
In the case of mutating the uORF located in the 5'-untranslated region of the gene encoding GDP-L-galactose phosphorylase, a key enzyme in the biosynthesis of the vital antioxidant ascorbic acid (AsA) in plant cells, the foliar AsA content in the lettuce mutants was increased by up to 150%, accompanied by significantly up-regulated tolerance to oxidative stress.
Through this work, it is established that CRISPR/Cas9 mediated editing is indeed an efficient and facile approach for rapidly introducing mutations to specific uORF targets, and that the resulting mutants are useful for the functional studies of both uORFs and their pORFs.
Different uORF mutants can confer differing effects on pORF translation. This permits a selection of the mutants with different protein accumulation levels for the genes under investigation, which is highly desirable for both basic and applied trait improvement research.
Because uORFs are widespread in eukaryotes, GAO Caixia said, it is possible that this approach will gain extensive applications in future genetic, genomic and biotechnological studies.
Financial support for this research is provided by the Ministry of Science and Technology of China, the Basic Science Center of the National Science Foundation of China, and Chinese Academy of Sciences.
Figure. Diagram of CRISPR/Cas9 mediated editing of a WT uORF that represses translation of downstream pORF mRNA. The mutant uorf reduces translation inhibition, thus leading to increased proteins production from the pORF. (Image by IGDB)
