Researchers from the University of Tokyo, MIT and Broad Institute of Harvard University, reveals a new killer bacteria Frances Cas9 (FnCas9) structure in the new study, and use this information FnCas9 structural transformation construct a variants. Research published in the journal Cell of February 25.
Professor, Institute of Basic Medical Sciences Medical Sciences University Osamu Nureki Jingdong, and Nureki laboratory assistant professor of biochemistry and biophysics Hiroshi Nishimasu is co-corresponding author of the paper. Broad Institute core member Zhang Feng is a co-author of the PhD thesis. February 2014, Zhang Feng had joined forces to collaborate with Professor Nureki generated CRISPR / Cas system is a key component of the composite --Cas9 first high-resolution images. This important research results published in the journal Cell.
RNA from CRISPR- Cas system boot enzyme Cas9 within DNA nucleic acid, can be combined with the double RNA guide sequence crRNA (CRISPR RNA) and tracrRNA (trans-activating RNA), or synthetic single guide RNA (sgRNA), cut with the guide RNA complementary the double-stranded target DNA. There are currently several Cas9 ortholog, such as Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) have been utilized to achieve Genome Editing in eukaryotic cells. Scientists are still committed to continue to improve these techniques.
Posted in November 2015 Nature Biotechnology journal on a research paper, the research team reported that a variant SaCas9 evolved, which can recognize a wider range of nucleotide sequences, targeting past CRISPR-Cas9 technology Unable to reach genomic loci.
December 2015, Zhang Feng led the MIT - Harvard Medical School and the Broad Institute of MIT McGovern Institute for Brain Research, design a revolutionary transformation of CRISPR-Cas9 gene editing system, greatly reducing the "off target "editing errors. The perfect technology to solve a major technical problem when using genome editing face. Research papers published in Science magazine.
In addition to requiring RNA-DNA pairing, Cas9 recognizes and cleaves DNA "PAM" (protospacer adjacent motif) also need to short DNA sequences present in the target DNA sequence adjacent a downstream, thereby limiting the Cas9 mediated gene targeting sequence can edit the group range. Cas9 orthologs from different microorganisms identify different PAM sequence, SpCas9 and SaCas9 respectively identify 5'-NGG-3 'and 5'-NNGRRT-3' PAMs.
Cas9 lineal length and sequence of homologues with a high degree of difference in the number of amino acid residues between about 900-1600, FnCas9 is one of the largest members. FnCas9 contains 1,629 amino acids, compared to other Cas9 ortholog as SpCas9 (1,368 amino acids) and SaCas9 (1,053 amino acids) is much greater. Notably, the previous study reported FnCas9 not only can mediate crRNA: tracrRNA dependent DNA cleavage, but also mediates scaRNA (small CRISPR / Cas related RNA): tracrRNA dependent regulation of gene expression. But the mechanism for FnCas9 performs its dual function remains unclear. In addition, the potential has yet to explore FnCas9 genome editing applications.
The researchers reported that won the crystal structure FnCas9-sgRNA- target DNA complexes in this new article in Cell, resolution 1.7 Å (Å). By comparing FnCas9 and other Cas9 orthologs SpCas9 and SaCas9, it reveals conserved features between distant kinship systems and CRISPR-Cas9 amazing structural differences. They found FnCas9 recognition is 5'-NGG-3 'PAM, and use this information to build a structure to identify a 5'-YG-3' PAM's Cas9 isomers. In addition, the researchers showed can be preassembled FnCas9-sgRNA ribonucleoprotein (RNP) complexes by microinjection into fertilized mouse eggs edit having 5'-YG-3 'PAM endogenous loci, thereby expanding the toolbox of targeted CRISPR-Cas9 space.
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