The method of efficient manipulation of mRNA shear is established by the cytosine monobase editing system based on CRISPR/Cas9.

The shearing of the mRNA precursor is an important process of gene transcription processing in higher organisms, and the traditional mRNA shear follows the "GU-AG" law, i.e. the main shear contains three conservative shear sites, namely the "GU" at the end of the inclusion 5' and the "AG" at the end of the 3' and the branch point "A" near the 3' end.
shears can be processed into one or more mature mRNAs by selecting one or more shearing sites, i.e. composition already and variable shears.
mRNA splicing plays a key role in gene expression regulation, and variable shears can increase protein diversity.
However, the current method of studying the function of mRNA shear or the function of mRNA-specific shear is still very limited, greatly limiting the functional study of a specific mRNA shear process.
, the Gao Caixia Research Group of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences used the model plant amoeba as a research material, and used the cytosine monobase editing system based on CRISPR/Cas9 to edit the shear conservative site "GU" at the end of the inclusion 5', and established an efficient method of manipulating mRNA shearing.
the study tested the method in four genes: AtHAB1, AtT30G6.16, AtRS31A and AtAct2, and found that the method was used to effectively remove or alter specific shear forms (Figure a).
the phenotype analysis found that the purification mutant hab1.1, which deleted HAB1.1, showed ABA-sensitive phenotype, consistent with the phenotype obtained by the previous person who expressed HAB1.2 in the HAB1 knockout mutant plant.
using this method, the researchers also found that the purified mutant rs31a.2, which removes RS31A.2, reduced sensitivity to fistapion in the wild compared to wild type (Figure b).
, the inclusion shear spot in the 5'UTR zone in AtAct2 is mutated by this method, and its constituent shear form can also be changed (Figure c).
graph: Using a single-base editing system to remove (a) or change a specific shear form (c), and prove that rs31a.2 mutants are less sensitive to fistapion (b) The previous generation used Cas9-mediated adenine monobase editing system to make the shear point "AG" at the end of the inclusion 3' mutation "GG", the mRNA precursor has a certain degree of reduction in the cutting efficiency at that point.
the study proves that Cas9-mediated cytosine monobase editing system can be efficiently removed or changed for specific shear forms.
the establishment of this method provides a powerful tool for studying the function of special shear form.
research paper Manipulating mRNA splicing by base editing in plants published online September 27 in The Journal of Science China Life Sciences (DOI: 10.1007/s11427-018-9392-7).
Gao Caixia Research Group master's student Xue Yanpin, associate researcher Zhang Huawei is the co-first author of the thesis.
the study was funded by the Ministry of Science and Technology, the Fund Committee and the Chinese Academy of Sciences.
Source: Institute of Genetics and Developmental Biology.