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CRISPR-Cas13a

CRISPR-Cas13a

The CRISPR-Cas13a, like other CRISPR-Cas systems functions in bacterial immunity but this system targets invading RNA rather than DNA. CRISPR-Cas13a is being engineered for RNA sensing applications such as live cell RNA tracking and detection of pathogenic viruses or bacteria.

Feng Zhang at the Broad Institute at MIT and Harvard was part of the team that originally showed that CRISPR-Cas9 gene editing could function in human cells . His lab has lead the developent of CRISPR-Cas13 as a biological research tool and for biomedical applications. Zhang’s lab has shown that one member of the Cas13 enzyme family, Cas13a from Leptotrichia wadei, can be used to knockdown mammalian and plant cell RNA to reduce the expression of the target gene . Zhang’s lab also engineered an inactive form of Cas13a with GFP that binds RNA without cutting which can make RNA of interest trackable in live cells .

Zhang and collaborators developed a nucleic acid detection system based on Cas13a, called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that can detect viruses, distinguish pathogenic bacteria, genotype human DNA and cell-free tumor DNA mutations . DNA or RNA is amplified with recombinase polymerase and converted to RNA with T7 RNA polymerase. When Cas13a recognizes its RNA target it cleaves non-target RNA nearby. SHERLOCK includes a reporter RNA that fluoresces when cleaved to show target virus sequence has been detected. Zhang collaborated with virology researcher Pardis Sabeti, also at the Broad Institute at MIT and Harvard to demonstrate the use of SHERLOCK to detect Zika virus and dengue virus, instrument-free, in patient body fluids (urine and saliva) at concentrations of 1 copy per microliter .

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