Establishing Programmable CRISPR/Cas13b-Mediated Knockdown System in Rice
Received date: 2024-10-06
Accepted date: 2024-12-17
Online published: 2025-04-14
CRISPR-Cas endonucleases mediate prokaryotic adaptive immunity by targeting foreign nucleic acids. CRISPR/Cas13b is a class 2 type VI-B ribonuclease that targets and cleaves single-stranded RNA. It exhibits higher RNA interference activity than Cas13a and Cas13c and causes fewer collateral effects than RxCas13d in mammalian cells. However, a programmable CRISPR/Cas13b-mediated RNA interference system for endogenous transcripts in rice has not yet been established. Here, we developed a CRISPR/Cas13b-mediated system to target endogenous transcripts in rice. Our CRISPR/Cas13b system could inhibit multiple endogenous mRNAs simultaneously. In addition, this system efficiently repressed endogenous long noncoding RNAs with more than 50% inhibition in stable transgenic plants. Furthermore, we found only weak collateral effects of the CRISPR/Cas13b-mediated system at the transcriptome-wide level, and no difference in the agronomic traits of stable transgenic rice in the field. We present a programmable CRISPR/Cas13b-mediated knockdown system for rice, offering a potential biotechnological tool for functional genomics and crop improvement.
Wang Shuman, Zhang Linqi, Gao Ruiren, Wei Guangbo, Dong Weiguo, Xu Jiming, Wang Zhiye . Establishing Programmable CRISPR/Cas13b-Mediated Knockdown System in Rice[J]. Rice Science, 2025 , 32(2) : 217 -227 . DOI: 10.1016/j.rsci.2024.12.012
| [1] | Abudayyeh O O, Gootenberg J S, Konermann S, et al. 2016. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 353: aaf5573. |
| [2] | Abudayyeh O O, Gootenberg J S, Essletzbichler P, et al. 2017. RNA targeting with CRISPR-Cas13. Nature, 550: 280-284. |
| [3] | Ai Y X, Liang D M, Wilusz J E. 2022. CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells. Nucleic Acids Res, 50(11): e65. |
| [4] | Aman R, Ali Z, Butt H, et al. 2018a. RNA virus interference via CRISPR/Cas13a system in plants. Genome Biol, 19: 1. |
| [5] | Aman R, Mahas A, Butt H, et al. 2018b. Engineering RNA virus interference via the CRISPR/Cas 13 machinery in Arabidopsis. Viruses, 10(12): 732. |
| [6] | Barrangou R, Fremaux C, Deveau H, et al. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315: 1709-1712. |
| [7] | Cox D B T, Gootenberg J S, Abudayyeh O O, et al. 2017. RNA editing with CRISPR-Cas13. Science, 358: 1019-1027. |
| [8] | East-Seletsky A, O’Connell M R, Knight S C, et al. 2016. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature, 538: 270-273. |
| [9] | Franco-Zorrilla J M, Valli A, Todesco M, et al. 2007. Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet, 39: 1033-1037. |
| [10] | Freije C A, Myhrvold C, Boehm C K, et al. 2019. Programmable inhibition and detection of RNA viruses using Cas13. Mol Cell, 76(5): 826-837.e11. |
| [11] | Gao C X. 2021. Genome engineering for crop improvement and future agriculture. Cell, 184(6): 1621-1635. |
| [12] | Guo M N, Ruan W Y, Li C Y, et al. 2015. Integrative comparison of the role of the PHOSPHATE RESPONSE1 subfamily in phosphate signaling and homeostasis in rice. Plant Physiol, 168(4): 1762-1776. |
| [13] | Hou X L, Wu P, Jiao F C, et al. 2005. Regulation of the expression of OsIPS1 and OsIPS2 in rice via systemic and local Pi signalling and hormones. Plant Cell Environ, 28(3): 353-364. |
| [14] | Hu Y P, Chen Y C, Xu J, et al. 2022. Metagenomic discovery of novel CRISPR-Cas13 systems. Cell Discov, 8(1): 107. |
| [15] | Kavuri N R, Ramasamy M, Qi Y P, et al. 2022. Applications of CRISPR/Cas13-based RNA editing in plants. Cells, 11(17): 2665. |
| [16] | Kelley C P, Haerle M C, Wang E T. 2022. Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR- Cas13d in mammalian cells. Cell Rep, 40(7): 111226. |
| [17] | Konermann S, Lotfy P, Brideau N J, et al. 2018. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell, 173(3): 665-676.e14. |
| [18] | Li B S, Sun C, Li J Y, et al. 2024. Targeted genome-modification tools and their advanced applications in crop breeding. Nat Rev Genet, 25(9): 603-622. |
| [19] | Ma X L, Zhang Q Y, Zhu Q L, et al. 2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 8(8): 1274-1284. |
| [20] | Mahas A, Aman R, Mahfouz M. 2019. CRISPR-Cas13d mediates robust RNA virus interference in plants. Genome Biol, 20(1): 263. |
| [21] | Miao Y S, Jiang L W. 2007. Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc, 2: 2348-2353. |
| [22] | Pickar-Oliver A, Gersbach C A. 2019. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol, 20(8): 490-507. |
| [23] | Sharma V K, Marla S, Zheng W G, et al. 2022. CRISPR guides induce gene silencing in plants in the absence of Cas. Genome Biol, 23: 6. |
| [24] | Sharp P M, Li W H. 1987. The codon adaptation index: A measure of directional synonymous Codon usage bias, and its potential applications. Nucleic Acids Res, 15(3): 1281-1295. |
| [25] | Shi P G, Murphy M R, Aparicio A O, et al. 2023. Collateral activity of the CRISPR/RfxCas13d system in human cells. Commun Biol, 6: 334. |
| [26] | Shmakov S, Abudayyeh O O, Makarova K S, et al. 2015. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell, 60(3): 385-397. |
| [27] | Smargon A A, Shi Y J, Yeo G W. 2020. RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering. Nat Cell Biol, 22(2): 143-150. |
| [28] | Tong H W, Huang J, Xiao Q Q, et al. 2023. High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat Biotechnol, 41: 108-119. |
| [29] | Wang Q X, Liu X, Zhou J H, et al. 2019. The CRISPR-Cas13a gene-editing system induces collateral cleavage of RNA in glioma cells. Adv Sci, 6(20): 1901299. |
| [30] | Wen W, Meinkotht J L, Tsien R Y, et al. 1995. Identification of a signal for rapid export of proteins from the nucleus. Cell, 82(3): 463-473. |
| [31] | Wiedenheft B, Sternberg S H, Doudna J A. 2012. RNA-guided genetic silencing systems in bacteria and archaea. Nature, 482: 331-338. |
| [32] | Wierzbicki A T, Blevins T, Swiezewski S. 2021. Long noncoding RNAs in plants. Annu Rev Plant Biol, 72: 245-271. |
| [33] | Wilson C, Chen P J, Miao Z, et al. 2020. Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nat Biotechnol, 38(12): 1431-1440. |
| [34] | Xu B B, Zhu Y D, Cao C C, et al. 2022. Recent advances in RNA structurome. Sci China Life Sci, 65(7): 1285-1324. |
| [35] | Xu C L, Zhou Y S, Xiao Q Q, et al. 2021. Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes. Nat Methods, 18(5): 499-506. |
| [36] | Yang H, Patel D J. 2024. Structures, mechanisms and applications of RNA-centric CRISPR-Cas13. Nat Chem Biol, 20: 673-688. |
| [37] | Yang L Z, Wang Y, Li S Q, et al. 2019. Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems. Mol Cell, 76(6): 981-997.e7. |
| [38] | Yu Y C, Pan Z Y, Wang X, et al. 2022. Targeting of SPCSV- RNase3 via CRISPR-Cas13 confers resistance against sweet potato virus disease. Mol Plant Pathol, 23(1): 104-117. |
| [39] | Zhan X H, Zhang F J, Zhong Z Y, et al. 2019. Generation of virus-resistant potato plants by RNA genome targeting. Plant Biotechnol J, 17(9): 1814-1822. |
| [40] | Zhang T, Zhao Y L, Ye J J, et al. 2019. Establishing CRISPR/ Cas13a immune system conferring RNA virus resistance in both dicot and monocot plants. Plant Biotechnol J, 17(7): 1185-1187. |
| [41] | Zhou J, Jiao F C, Wu Z C, et al. 2008. OsPHR2 is involved in phosphate- starvation signaling and excessive phosphate accumulation in shoots of plants. Plant Physiol, 146(4): 1673-1686. |
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