CRISPR-Cas13 is an RNA targeting and editing system based on the bacterial immune system that protects them from viruses. Cas13, previously known as C2c2, is the effector protein that targets and cleaves invading nucleic acids from viruses in type VI CRISPR-Cas systems. The CRISPR-Cas13 system is analogous to the CRISPR-Cas9 system. However, unlike Cas-9 which targets DNA, Cas-13 targets and cleaves single stranded RNA. Cas-13 was first discovered in L. shahii, a species of the Leptotrichia bacteria while researchers were looking for previously unidentified CRISPR systems.
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 CRIPR-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 .
The RNA Editing for Programmable A to I Replacement (REPAIR) system based on CRISPR-Cas13 was developed by Zhang’s lab to edit RNA transcripts with pathogenic mutations . Here, the Cas13b ortholog from the bacterium Prevotella sp. P5–125 was engineered to not cleave RNA (dCas13b) and fused to an adensosine deaminase enzyme which changes A to I in targeted spot on an RNA transcript as determined by the user supplied guide RNA. Zhang’s team used REPAIR to correct RNA transcripts in cell lines from patients with mutations causing Fanconi anemia and X-linked Nephrogenic diabetes insipidus. The REPAIR system is engineered for viral delivery into cells. There are less sequence constraints for CRISPR-Cas13 RNA editing than for CRISPR-Cas9 DNA editing, making it more flexible. However since the REPAIR system targets RNA not DNA, effects are temporary. Zheng's group suggests it would be suitable to treat diseases with acute phases like inflammation or modify proteins in the disease pathway to slow disease progression.
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 .
Paper published on in vivo RNA targeting
Pardis C. Sabeti
C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector
Abudayyeh et al.
CRISPR/Cas13 as a Tool for RNA Interference
New Version of CRISPR, Developed by Feng Zhang–Led Team, Can Target and Edit RNA
RNA editing with CRISPR-Cas13
David B. T. Cox
Documentaries, videos and podcasts
- CRISPRClustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune response that provides immunity against foreign nucleic acids, such as viral DNA and bacterial plasmids, through the use of crRNAs (CRISPR RNAs) and associated Cas genes.
- CRISPR/Cas ToolsGene-editing technique that has revolutionized the field of genetic engineering
- Cluster: Synthetic biologyA cluster of topics related to synthetic biology.
- Synthetic biologyInterdisciplinary branch of biology and engineering, applying multiple disciplines to build artificial biological systems for research, engineering, and medical applications.
- CRISPR-Cas12aCRISPR-Cas12a is is a genome editing tool similar to CRISPR-Cas9. CRISPR-Cas12a is a set of RNA guided DNA targeting proteins capable of producing targeted double-stranded DNA (dsDNA) breaks, targeted single-stranded DNA breaks, and indiscriminate ssDNA degradation in trans. The effector protein Cas12a is also known at Cpf1.
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- Gene editing
- RNARNA stands for ribonucleic acid. Messenger RNA (mRNA) is copied or transcribed from DNA and carries the coding information for the generation of a protein encoded by a gene. Different types of non-coding RNAs have roles in converting mRNA into protein and perform other regulatory functions in the cell.
- Trans-activating crRNA
- Gene knockdown