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Recently, Cas13 has been shown to be able to target RNA instead of DNA for editing. Established members of the Cas13 protein family include Cas13a (formerly known as C2c2), Cas13b, and Cas13c. In 2017, Abudayyeh and colleagues demonstrated that CRISPR/Cas13 could be a flexible platform for studying RNA in mammalian cells. This represents that Cas13 is a potentially safe alternative to Cas9 because it induces a loss of functioning phenotype without causing loss of the target gene sequence.
Two distinct RNase activities of CRISPR/Cas13
Unlike most dsDNA-targeted CRISPR systems, Cas13 has two advanced eukaryotic and prokaryotic nucleotide-binding (HEPN) domains that target single-stranded RNA (ssRNA). Together they form a ribonuclease active site, enabling it to act as an RNA-guided RRNA endonuclease (as a single-stranded RNA (ssRNA) substrate). In addition, studies show that Cas13 has a unique ribonuclease activity responsible for CRISPR RNA maturation, which is different from its RNA-activated ssRNA degradation activity. The functions of these double ribonucleases differ chemically and mechanically from each other. The two ribonuclease activities of Cas13 enable multiplex processing and direct RNA loading, allowing sensitive cell transcript detection.
Figure 1: Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities. (Alexandra East-Seletsky. 2016)
Application of Cas13 for Nucleic Acid Detection and Targeting
Gootenberg, etc. used Cas13a, a CRISPR effector that targets RNA, to develop a Cas13a-based molecular detection platform called SHERLOCK (specific high-sensitivity enzyme reporter gene unlock) for in vitro detection of DNA and RNA with a single-base mismatch specificity and attomolar sensitivity.
Cameron Myhrvold's research shows that Cas13a-based SHERLOCK can detect any virus, virus resistance mutations, and clinically relevant virus single nucleotide polymorphisms in body fluids.
SHERLOCK can detect nucleic acids in patients with serum or urine at low to low Armole concentrations, which can detect tumor mutations in cell-free DNA (cfDNA).
In addition, nuclease-inactivated dCas13, which appears as an RNA-directed RNA-binding protein without endonuclease activity, can be fused with spectrally separated fluorescent proteins to enable visualization of endogenous RNA and its transport in living cells. This method is undoubtedly a cutting-edge tool suitable for monitoring the characteristics of transcript localization and related regulatory pathways in real-time, as well as specific molecular mechanisms associated with well-characterized mRNAs.
CRISPR/Cas13 and gene therapy
Figure 2: Therapeutic strategies based on CRISPR/Cas13-based tools. (Panayiota Papasavva. 2019)
To date, most research has focused on type II Cas9 proteins. However, much remains to be discovered about the role of Cas13 in biomedical and disease treatment.
CRISPR/Cas9 PlatformCB is one of the leading biotechnology companies dedicated to providing customers with CRISPR system solutions to promote scientific research, drug development, disease treatment research, and more. Our scientists have deep pharmacological knowledge and rich experience in experimental operation and data processing. Whether you need sgRNA design and synthesis or Cas13 vector construction, our trained experts will support you with their strong problem-solving capabilities and expertise. Contact us to learn more about our services. Looking forward to working with you in the future.