RNA Pol III promoters—key players in precisely targeted plant genome editing

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.

Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern

“…The core competent for CRISPR/Cas9 system contains the expression cassettes of sgRNA and the SpCas9 nuclease. Guide RNAs for genome editing have been produced using a range of Pol III promoters (Xie et al, 2015; Kor et al, 2022). We found seven U6 small nuclear ribonucleoprotein genes ( Ceric.17G074700, Ceric.33G040100, Ceric.09G088700, Ceric.02G026900, Ceric.1Z290000, Ceric.03G070800, Ceric.03G071600 ) in the C. richardii genome (https://phytozome-next.jgi.doe.gov/info/Crichardii_v2_1), which showed high expression in gametophyte, leaf, stem, and root (Supplemental Figure S1A).…”

“…We found seven U6 small nuclear ribonucleoprotein genes ( Ceric.17G074700, Ceric.33G040100, Ceric.09G088700, Ceric.02G026900, Ceric.1Z290000, Ceric.03G070800, Ceric.03G071600 ) in the C. richardii genome (https://phytozome-next.jgi.doe.gov/info/Crichardii_v2_1), which showed high expression in gametophyte, leaf, stem, and root (Supplemental Figure S1A). However, the promoters of these C. richardii genes do not contain the upstream sequence element (USE) and TATA elements, which are the typical structural properties of the Pol III promoters (Kor et al, 2022). Therefore, we used the sequences of the A. thaliana U6-26 snRNA (X52528, AT3G13857) and the T. aestivum U6 gene (X52528, ENSRNA050022746-T1) (Poovaiah et al, 2021) sequences to compare with the upstream U6 promoter regions in C. richardii .…”

“…This system can edit genes with high efficiency in C. richardii based on the success with CrSAL1, CrPDS and other genes. In most of the CRISPR/Cas9 constructs, the RNA polymerase III-type 3 - U3 or U6 promoters are employed for expression of sgRNA in monocots, eudicots, gymnosperms, and bryophytes (Kor et al, 2022). Although we did not use CrU6 promoters due its low expression, we hypothesize that they can have potential applications in the genome editing of ferns.…”

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

“…In addition, Cas9 expression in germ cells (oocytes, daughter cells, and early embryos) leads to heritable editing and reduces somatic mutations in organogenic plants. Efficient CRISPR/Cas9 systems based on germline-specific promoters may reduce chimerism and thus reduce the workload for the characterization of edited plants [ 170 , 171 , 172 ].…”

Optimization of CRISPR-Cas9 system in Eustoma grandiflorum

“…Additionally, RNA transcribed by Pol III promoters is usually not capped or polyadenylated, allowing the transcription product to function immediately in the cell. 21 , 22 The U6 promoter uses G as the transcription initiation site. Many studies have shown that the efficiency of a U6 promoter is substantially degraded when used in a distantly related species.…”