New gene editing tool that can act the role of Cas9
Researchers from Hebei University of Technology and Zhejiang University School of Medicine report the successful application of Argonaute of Natronobacterium gregoryi to achieve genomic DNA editing. These innovative research results was published on May 2nd in the journal Nature Biotechnology.
Cas9 endonucleolytic RNA endonuclease guided genome is the most common editing tools, which use the RNA-DNA hybrid to specifically cleaves the genomic sequence. Although researchers have managed to transform several Cas9 system to improve its efficiency and specificity, its sequence Wizard - Wizard of tolerance and target RNA sequence mismatches relatively easy to form secondary structure limits the usefulness of the system Cas9-sgRNA.
The study was led by associate professor Dr.Chunyu Han at the Department of Biological Sciences, College of Life Science and Engineering, Hebei University of Science. Argonautes is a endonuclease family, which utilizes 5 phosphorylated short single-stranded nucleic acid as a guide to cut the target sequence. Unlike Cas9 which exists only in prokaryotes, Argonautes were conserved during evolution and present in almost all organisms. Although most Argonautes play an important role in RNA silencing by binding single-stranded (ss) RNAs, some Argonautes are combined with ssDNAs to cleave the target sequences. Precise Cas9 binding requires a guide RNA to form the secondary RNA hybrid structure and can only cut the target sequence upstream of PAM. Argonaute, on the other side, does not require the presence of 3’ RNA-RNA structure and its functions are not limited to certain sequence-specific target sites.
In this Nature Biotechnology paper, the authors reported that the Natronobacterium gregoryiArgonaute (NgAgo) is a DNA-guided endonuclease suitable for genome editing in human cells. NgAgo can be combined with a 5 'phosphorylated single-stranded Wizard DNA (gDNA) of only 24 nucleotides in length, to efficiently generate site-specific DNA double-strand breaks. The NgAgo–gDNA system does not require a protospacer-adjacent motif (PAM), as does Cas9, and preliminary characterization suggests a low tolerance to guide–target mismatches and high efficiency in editing (G+C)-rich genomic targets.Thus, NgAgo-gDNA system is a precise and effective tool that can implement genome editing in mammalian cells.
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