Efficient plant single base editing system nCas9-PBE.

Single nucleotide point mutation is the genetic basis for the variation of many important agrotics in crops.
variation of mono-bases can lead to amino acid replacement or protein translation termination, which alters gene function, potentially producing excellent allegiants and excellent character.
traditional mutagenic and single-base mutation screening techniques, such as TILLING, require genome-scale screening, which is time-consuming, labor-consuming, and identifies a limited number and variety of point mutations.
Genomic editing technology, especially those based on crispr/Cas9 systems, can produce DNA double strand fracture (DSB) at genomic target site, using human-provided exogenetic supply DNA as a template for achieving single base mutations to target genes through HR-mediated DNA repair.
, the effernation efficiency of esophageal recombination in plants is currently very low, and it is difficult to achieve efficient and stable single-base mutations.
, plant breeding and gene function research urgently need new technologies to improve the efficiency of genome single base fixed-point mutations in order to achieve targeted improvement of gene function and agrotic features.
The Gao Caixia Research Group of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences is devoted to the research and application of crop genome editing methods.
based on the previous work, drawing on mammalian single base editing methods, Gao Caixia Research Group used Cas9 variants (nCas9-D10A) to fuse rat cytosine deaminase (rAPOBEC1) and UGI, an efficient plant monobase editing system nCas9-PBE, successfully achieves efficient and accurate single base-based mutations in the genomes of three important crops (wheat, rice and corn).
results were presented on October 31, 2016, subject to rigorous peer review, and were officially accepted on February 5, 2017 and published online February 27 in the journal Nature Biotechnology (doi:10.1038/nbt.3811).
Through a detailed analysis of the results of seven-bit mutations of the reported gene BFP and five endogenetic genes in three crops in the progenitor body, it was found that nCas9-PBE could achieve C-to-T replacement of target DNA, C base The ammonia window covers 7 nucleotides in the target sequence (3-9 bits from the far end of PAM), where the replacement efficiency of a single C is 0.39-7.07% and the replacement efficiency of multiple Cs is as high as 12.48%.
through genetic transformation, the system obtained wheat, rice and corn mutant plants replaced by single base in the target area, with mutation efficiency of up to 43.48%.
nCas9-PBE technology does not require DNA double-stranded fracture (DSB) at the target point of the genome, nor does it require the involvement of the body DNA, and is simple, wide-ranging and efficient.
the successful establishment and application of the nCas9-PBE single base editing system provides a reliable solution for the efficient and large-scale creation of single base mutants, and provides important technical support for crop genetic improvement and new variety cultivation.
And Wang Yanpeng, Ph.D. students from Gao Caixia Research Group, are the co-authors of the paper.
the study was supported by the Ministry of Science and Technology, the Ministry of Agriculture, the Chinese Academy of Sciences and the National Natural Resources Fund.
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