RNA Pol III Promoters-key Players in Precisely Targeted Plant Genome Editing

Overview

Journal Front Genet

Date 2023 Jan 23

PMID 36685866

Authors

Sakshi Dharmendra Kor

Naimisha Chowdhury

Ajay Kumar Keot

Kalenahalli Yogendra

Channakeshavaiah Chikkaputtaiah

Palakolanu Sudhakar Reddy

Affiliations

Soon will be listed here.

Abstract

The clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein Cas) system is a powerful and highly precise gene-editing tool in basic and applied research for crop improvement programs. CRISPR/Cas tool is being extensively used in plants to improve crop yield, quality, and nutritional value and make them tolerant to environmental stresses. CRISPR/Cas system consists of a Cas protein with DNA endonuclease activity and one CRISPR RNA transcript that is processed to form one or several short guide RNAs that direct Cas9 to the target DNA sequence. The expression levels of Cas proteins and gRNAs significantly influence the editing efficiency of CRISPR/Cas-mediated genome editing. This review focuses on insights into RNA Pol III promoters and their types that govern the expression levels of sgRNA in the CRISPR/Cas system. We discussed Pol III promoters structural and functional characteristics and their comparison with Pol II promoters. Further, the use of synthetic promoters to increase the targeting efficiency and overcome the structural, functional, and expressional limitations of RNA Pol III promoters has been discussed. Our review reports various studies that illustrate the use of endogenous U6/U3 promoters for improving editing efficiency in plants and the applicative approach of species-specific RNA pol III promoters for genome editing in model crops like Arabidopsis and tobacco, cereals, legumes, oilseed, and horticultural crops. We further highlight the significance of optimizing these species-specific promoters' systematic identification and validation for crop improvement and biotic and abiotic stress tolerance through CRISPR/Cas mediated genome editing.

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References

Khan M, Hu L, Zhu M, Zhai Y, Khan S, Ahmar S . Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production. J Cell Physiol. 2020; 236(3):1996-2007. DOI: 10.1002/jcp.29986. View

Wang S, Zhang S, Wang W, Xiong X, Meng F, Cui X . Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Rep. 2015; 34(9):1473-6. DOI: 10.1007/s00299-015-1816-7. View

Zhang Z, Hua L, Gupta A, Tricoli D, Edwards K, Yang B . Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing. Plant Biotechnol J. 2019; 17(8):1623-1635. PMC: 6662106. DOI: 10.1111/pbi.13088. View

Bogenhagen D, Wormington W, Brown D . Stable transcription complexes of Xenopus 5S RNA genes: a means to maintain the differentiated state. Cell. 1982; 28(2):413-21. DOI: 10.1016/0092-8674(82)90359-2. View

Ueta R, Abe C, Watanabe T, Sugano S, Ishihara R, Ezura H . Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. Sci Rep. 2017; 7(1):507. PMC: 5428692. DOI: 10.1038/s41598-017-00501-4. View

Jiang W, Henry I, Lynagh P, Comai L, Cahoon E, Weeks D . Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing. Plant Biotechnol J. 2016; 15(5):648-657. PMC: 5399004. DOI: 10.1111/pbi.12663. View

Lobo S, Hernandez N . A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. Cell. 1989; 58(1):55-67. DOI: 10.1016/0092-8674(89)90402-9. View

Char S, Wei J, Mu Q, Li X, Zhang Z, Yu J . An Agrobacterium-delivered CRISPR/Cas9 system for targeted mutagenesis in sorghum. Plant Biotechnol J. 2019; 18(2):319-321. PMC: 6953201. DOI: 10.1111/pbi.13229. View

Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H . Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep. 2015; 5:10342. PMC: 4448504. DOI: 10.1038/srep10342. View

10.

Gao Z, Herrera-Carrillo E, Berkhout B . RNA Polymerase II Activity of Type 3 Pol III Promoters. Mol Ther Nucleic Acids. 2018; 12:135-145. PMC: 6023835. DOI: 10.1016/j.omtn.2018.05.001. View

11.

Fowlkes D, Shenk T . Transcriptional control regions of the adenovirus VAI RNA gene. Cell. 1980; 22(2 Pt 2):405-13. DOI: 10.1016/0092-8674(80)90351-7. View

12.

Braatz J, Harloff H, Mascher M, Stein N, Himmelbach A, Jung C . CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape (). Plant Physiol. 2017; 174(2):935-942. PMC: 5462057. DOI: 10.1104/pp.17.00426. View

13.

Richard P, Manley J . Transcription termination by nuclear RNA polymerases. Genes Dev. 2009; 23(11):1247-69. PMC: 2763537. DOI: 10.1101/gad.1792809. View

14.

Filipowicz W, Kiss T, Marshallsay C, Waibel F . U-snRNA genes, U-snRNAs and U-snRNPs of higher plants. Mol Biol Rep. 1990; 14(2-3):125-9. DOI: 10.1007/BF00360443. View

15.

Waibel F, Filipowicz W . U6 snRNA genes of Arabidopsis are transcribed by RNA polymerase III but contain the same two upstream promoter elements as RNA polymerase II-transcribed U-snRNA genes. Nucleic Acids Res. 1990; 18(12):3451-8. PMC: 330996. DOI: 10.1093/nar/18.12.3451. View

16.

Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X . CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol. 2014; 87(1-2):99-110. DOI: 10.1007/s11103-014-0263-0. View

17.

Nie L, Das Thakur M, Wang Y, Su Q, Zhao Y, Feng Y . Regulation of U6 promoter activity by transcriptional interference in viral vector-based RNAi. Genomics Proteomics Bioinformatics. 2010; 8(3):170-9. PMC: 5054135. DOI: 10.1016/S1672-0229(10)60019-8. View

18.

Kui L, Chen H, Zhang W, He S, Xiong Z, Zhang Y . Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, . Front Plant Sci. 2017; 7:2036. PMC: 5226938. DOI: 10.3389/fpls.2016.02036. View

19.

Huang B, Zhuo R, Fan H, Wang Y, Xu J, Jin K . An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (). Front Plant Sci. 2022; 13:822022. PMC: 8874139. DOI: 10.3389/fpls.2022.822022. View

20.

Huang Y, Wang Y, Zeng B, Liu Z, Xu X, Meng Q . Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella. Insect Biochem Mol Biol. 2017; 89:71-78. DOI: 10.1016/j.ibmb.2017.08.009. View