Biology:Cas13
Cas13 is a CRISPR-associated enzyme that targets RNA. Unlike the DNA-targeting Cas9, Cas13 utilizes a single RNA-guided endonuclease to bind and cleave specific RNA sequences. It employs two distinct ribonuclease activities: one for processing its own CRISPR RNA (crRNA) and another for degrading the target RNA.[1][2][3]
The system's specificity allows for the correction of mutations at the transcript level. For example, it has been used to repair KRAS-G12D mRNA in pancreatic cancer models with high efficiency while minimizing effects on healthy cells.[1] It has been adapted into tools such as the REPAIR platform, which edits RNA bases to treat genetic disorders, including Usher syndrome, in animal models. Cas13 also possesses collateral RNA-cleavage activity, which is utilized in diagnostic platforms like SHERLOCK to detect pathogens, tumor DNA, and viral variants with high sensitivity.[3] Its PAM-independent targeting and reduced off-target effects make it suitable for RNA imaging, phage genome engineering, and transient gene regulation.[2]
History
In 2016, researchers in Feng Zhang's group at MIT and the Broad Institute characterized the nuclease Cas13a (formerly C2c2) from the bacterium Leptotrichia shahii.[4] Its collateral cleavage property is central to several diagnostic technologies.[5][6][7]
In 2018, a team led by Silvana Konermann and Patrick Hsu at the Salk Institute identified Cas13d, a compact subclass of RNA-targeting CRISPR effectors. An engineered variant of Ruminococcus flavefaciens Cas13d, named CasRx, demonstrated high efficiency and specificity in human cells compared to RNA interference. CasRx can be packaged into adeno-associated virus (AAV) vectors for transcriptome engineering and gene therapy.[8]
In 2021, researchers characterized miniature Cas13 protein variants, Cas13X and Cas13Y. Studies using the SARS-CoV-2 N gene sequence as a target showed that mCas13, when coupled with RT-LAMP, detected SARS-CoV-2 in synthetic and clinical samples with high sensitivity and specificity, comparable to RT-qPCR.[9]
Applications
Cas13 has been adapted to function as an RNA editor capable of correcting mutations without modifying DNA. The REPAIR system utilizes a catalytically inactive Cas13 (dCas13) that binds target RNA without cleaving it. This dCas13 is fused to the catalytic domain of ADAR2, an enzyme that converts adenosine (A) to inosine (I), which is interpreted by the cellular machinery as guanosine (G). This complex can be guided to specific mRNA locations to correct disease-causing mutations.[10]
When combined with a high-fidelity ADAR2 variant, the REPAIR system has demonstrated the ability to edit targets with minimal off-target effects. In murine models of Usher syndrome, the dCas13–ADAR system, delivered via viral vectors, restored usherin protein levels, corrected defective transcripts, and improved vision. These results indicate that Cas13-mediated RNA editing may offer a viable approach for treating genetic disorders.[11]
Further refinements have led to the development of "dead" Cas13b, which retains binding capabilities but lacks cleavage activity. Paired with a guide RNA that includes a specific A-to-C mismatch at the target site, this system directs the ADAR2 enzyme to edit a single base. Initial tests in human cells showed reliable editing within a 30-nucleotide window with significant precision.[12]
See also
References
- ↑ 1.0 1.1 "CRISPR-Cas13a system: a novel approach to precision oncology". Cancer Biology & Medicine 17 (1): 6–8. February 2020. doi:10.20892/j.issn.2095-3941.2019.0325. PMID 32296572.
- ↑ 2.0 2.1 "Advances in application of CRISPR-Cas13a system". Frontiers in Cellular and Infection Microbiology 14. 2024. doi:10.3389/fcimb.2024.1291557. PMID 38524179.
- ↑ 3.0 3.1 "CRISPR-Cas13a system: A novel tool for molecular diagnostics". Frontiers in Microbiology 13. 2022. doi:10.3389/fmicb.2022.1060947. PMID 36569102.
- ↑ "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector". Science 353 (6299). August 2016. doi:10.1126/science.aaf5573. PMID 27256883.
- ↑ "Nucleic acid detection with CRISPR-Cas13a/C2c2". Science 356 (6336): 438–442. April 2017. doi:10.1126/science.aam9321. PMID 28408723. Bibcode: 2017Sci...356..438G.
- ↑ "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6". Science 360 (6387): 439–444. April 2018. doi:10.1126/science.aaq0179. PMID 29449508. Bibcode: 2018Sci...360..439G.
- ↑ "SPRINT: a Cas13a-based platform for detection of small molecules". Nucleic Acids Research 48 (17): e101. September 2020. doi:10.1093/nar/gkaa673. PMID 32797156.
- ↑ "Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors". Cell 173 (3): 665–676.e14. April 2018. doi:10.1016/j.cell.2018.02.033. PMID 29551272.
- ↑ "A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics". ACS Synthetic Biology 10 (10): 2541–2551. October 2021. doi:10.1021/acssynbio.1c00181. PMID 34546709.
- ↑ "RNA editing with CRISPR-Cas13". Science (New York, N.Y.) 358 (6366): 1019–1027. 2017-11-24. doi:10.1126/science.aaq0180. PMID 29070703. Bibcode: 2017Sci...358.1019C.
- ↑ "Comparison of CRISPR-Cas13b RNA base editing approaches for USH2A-associated inherited retinal degeneration" (in en). Communications Biology 8 (1): 200. 2025-02-08. doi:10.1038/s42003-025-07557-3. ISSN 2399-3642. PMID 39922978.
- ↑ "Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors" (in English). Cell 173 (3): 665–676.e14. 2018-04-19. doi:10.1016/j.cell.2018.02.033. ISSN 0092-8674. PMID 29551272.
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