The new multi-nucleotide target ingestion system AFIDs provide a powerful genome editing tool for breeding.

The plant genome has a wide variety of regulatory elements, functional base sequences, and non-coding DNA, such as promoter-shun acting elements, miRNA coding sequences, and intergene regions with regulatory functions.
these DNA sequences play an important role in regulating gene expression, transcription translation and so on, and are also the key target areas for gene function research and genetic improvement.
crispR/Cas9-based genome editing technology has been widely used in functional genetic research and crop genetic improvement.
The SgRNA-guided Cas9 nucleic acid enzyme can produce DNA double-stranded fracture (DSB) at the genomic target site, and cells are repaired by non-hoe-end connection (NHEJ), often prone to the insertion or deletion of 1 to 3 nucleotides.
However, this insertion/missing mutation is difficult to effectively disrupt the function of regulating DNA, so the classic CRISPR/Cas9 cannot effectively manipulate the above important DNA functional elements. Based on this
, the development of a new, accurate and predictable polynucleotide deletion genome editing system is of great significance to the functional analysis and application of regulating DNA sequences.
the Gao Caixia Research Group of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences has long been committed to the research and development of new technologies for plant genome editing.
recently, the team has for the first time combined wild-type SpCas9 with cytosine deaminase APOBEC, ulysine-based deaminosase (UDG) and aplyagnin-site lyase (AP lyase) based on the principle of cytosine desiamin and base-cut remediation (BER), and has successfully achieved a new type of polynucleotide target removal system (AFIDs) in rice and wheat.
considering the presence of the BER system in plant cells themselves, the team first constructed three forms of AFID system (AFID-1 to 3) using a highly desation-active APOBEC3A deaminase, and tested several endogenous DNA targets in rice and wheat cells, resulting in a removal efficiency of up to 33.1% of AFID-3-mediated, and producing a high proportion of multi-nucleotide removal from different 5'cytosine to Cas9 cutting points.
the researchers further screened the non-cosyd deaminase and found that the truncated APOBEC3B deaminase (A3Bctd) not only had higher desoritamine activity, but also had a narrower desamine window.
researchers replaced A3Bctd with A3A in the AFID-3 system, thus developing the eAFID-3 system.
eAFID-3 more efficiently mediates predictable deletions from desperation points in preference for TC base sequence to Cas9 cutting sites, 1.5 times more efficient than AFID-3. In addition, the
, the team used the AFID-3 system to target the effect subbinding elements on the osSWEET14 gene promoter of rice, and obtained the mutant plant strain of polynucleotide deletion.
found that the multi-nucleotide removal of rice mutants produced by the system was more resistant to white leaf dead bacteria than the insertion loss of 1 to 2 bp.
AFID system sits accurately and predictable lying at plant brokering.
(a) diagram of how the AFID system works.
(b) different forms of construction of AFID systems (AFID-1 to 3, eAFID-3).
(c) a predictable deletion ratio comparison between AFID-3 and Cas9. comparison of predictable deletion types
(d) AFID-3 and eAFID-3.
(e) AFID-3-mediated rice can predict the difference in the resistance of polynucleotide deletion mutants to 1 to 2 bp insertion missing mutations in white leaf disease.
AFID system has the advantages of high efficiency, precision and predictability, so the establishment of the system can provide a powerful genome editing tool for the functional research and design breeding of plant genome regulation DNA.
published the findings online June 29 in Nature Biotechnology (DOI: 10.1038/s41587-020-0566-4).
Gao Caixia Research Group postdoctoral wang Shengxing, postdoctoral Zong Yu, doctoral student Lin Qiupeng and associate researcher Zhang Huawei are the co-first authors of the thesis, Gao Caixia is the communication author of the paper. Qiu Jinlong Research Group of the Institute of Microbiology of the Chinese Academy of Sciences
also participated in the research work.
the research was supported by the Chinese Academy of Sciences' strategic leading science and technology project, the national special project of major science and technology of genetically modified, and the project of the National Natural Science Foundation of China.
Source: Institute of Genetics and Developmental Biology.