Cas13

Last updated

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]

Contents

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. 1 2 Zhang J, You Y (February 2020). "CRISPR-Cas13a system: a novel approach to precision oncology". Cancer Biology & Medicine. 17 (1): 6–8. doi:10.20892/j.issn.2095-3941.2019.0325. PMC   7142841 . PMID   32296572.
  2. 1 2 Zhang Y, Li S, Li R, Qiu X, Fan T, Wang B, Zhang B, Zhang L (2024). "Advances in application of CRISPR-Cas13a system". Frontiers in Cellular and Infection Microbiology. 14 1291557. doi: 10.3389/fcimb.2024.1291557 . PMC   10958658 . PMID   38524179.
  3. 1 2 Zhao L, Qiu M, Li X, Yang J, Li J (2022). "CRISPR-Cas13a system: A novel tool for molecular diagnostics". Frontiers in Microbiology. 13 1060947. doi: 10.3389/fmicb.2022.1060947 . PMC   9772028 . PMID   36569102.
  4. ↑ Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F (August 2016). "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector". Science. 353 (6299) aaf5573. doi:10.1126/science.aaf5573. PMC   5127784 . PMID   27256883.
  5. ↑ Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F (April 2017). "Nucleic acid detection with CRISPR-Cas13a/C2c2". Science. 356 (6336): 438–442. Bibcode:2017Sci...356..438G. doi:10.1126/science.aam9321. PMC   5526198 . PMID   28408723.
  6. ↑ Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F (April 2018). "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6". Science. 360 (6387): 439–444. Bibcode:2018Sci...360..439G. doi:10.1126/science.aaq0179. PMC   5961727 . PMID   29449508.
  7. ↑ Iwasaki RS, Batey RT (September 2020). "SPRINT: a Cas13a-based platform for detection of small molecules". Nucleic Acids Research. 48 (17): e101. doi: 10.1093/nar/gkaa673 . PMC   7515716 . PMID   32797156.
  8. ↑ Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD (April 2018). "Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors". Cell. 173 (3): 665–676.e14. doi:10.1016/j.cell.2018.02.033. PMC   5910255 . PMID   29551272.
  9. ↑ Mahas A, Wang Q, Marsic T, Mahfouz MM (October 2021). "A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics". ACS Synthetic Biology. 10 (10): 2541–2551. doi:10.1021/acssynbio.1c00181. PMC   8482783 . PMID   34546709.
  10. ↑ Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F, Cox DB (2017-11-24). "RNA editing with CRISPR-Cas13". Science. 358 (6366). New York, N.Y.: 1019–1027. Bibcode:2017Sci...358.1019C. doi:10.1126/science.aaq0180. PMC   5793859 . PMID   29070703.
  11. ↑ Major L, Salman A, McDermott LA, Yang J, King AJ, McClements ME, MacLaren RE, Fry LE (2025-02-08). "Comparison of CRISPR-Cas13b RNA base editing approaches for USH2A-associated inherited retinal degeneration". Communications Biology. 8 (1): 200. doi:10.1038/s42003-025-07557-3. ISSN   2399-3642. PMC   11807095 . PMID   39922978.
  12. ↑ Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD, Konermann S (2018-04-19). "Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors". Cell. 173 (3): 665–676.e14. doi:10.1016/j.cell.2018.02.033. ISSN   0092-8674. PMC   5910255 . PMID   29551272.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.