Research team from the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences (CAS) develop a predictable multi-nucleotide deletion system in plants.
Besides conventional coding DNA that translates into a protein product, non-coding nucleic acids such as small regulatory elements are essential and play key roles in regulation of gene expression, transcription and translation. MicroRNAs (miRNA) is one type of such regulatory elements that bind to miRNA binding sites and cis-acting elements which are only five to 24 nucleotides long, these sites are promising targets for gene function studies.
CRISPR-Cas9 a widely used genome engineering system, has been acknowledged in the scientific community as a major breakthrough in genetic engineering. This system however, has its shortcomings which result in frequent short insertions and deletions of one to three base pairs. The inconsistency of these small sections results in technical difficulty in disrupting these regulatory DNA strands. This warranted the development of a precise, predictable multi-nucleotide deletion system.
The research team led by Professor Gao Caixia has been focussing on developing novel technologies to achieve efficient and specific genome engineering. Making use of the cytidine deamination and base excision repair (BER) mechanism, the researchers were able to formulate a new series of systems that combines combine Cas9 with human APOBEC3A (A3A), uracil DNA-glucosidase (UDG) and AP lyase.
The series of APOBEC-Cas9 fusion-induced deletion systems (AFIDs) showed to successfully resulted in specific and predictable multi-nucleotide deletions in rice and wheat genomes.
“AFID-3 produced a variety of predictable deletions extending from the 5’-deaminated Cs to the Cas9 cleavage sites, with the average predicted proportions over 30 percent,” said Professor Gao.
The researchers further screened the deamination activity of different cytosine deaminases in rice protoplasts, and found that the truncated APOBEC3B (A3Bctd) displayed not only a higher base-editing efficiency but also a narrower window than other deaminases.
They were able to produce more uniform deletions after replacing A3A with the truncated cytidine deaminase to generate eAFID-3.
Moreover, the researchers used the AFID system to target the effector-binding element of OsSWEET14 in rice, and found that the predictable deletion mutants conferred enhanced resistance to rice bacterial blight.
AFID systems have shown to be an improvement to other current tools for generating predictable multi-nucleotide targeted deletions within the protospacer, providing promise of a more robust deletion tools for basic research and genetic improvement.
The team’s scientific paper, entitled “Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9,” was published in Nature Biotechnology. [APBN]