Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-Based Transcriptional Activation

Nor Akmar Abdullah, Siti; Abdullah, Akmar; Mayes, Sean

Abstract

Background:
CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely utilised as a gene activation system exploiting its ability in recruiting various transcriptional activation domains (TADs) for enhancing expression of the target gene(s). Drought tolerant and resource efficient crops like millets have potential for mitigating effects of climate change and for enhancing food security.

Objective:
This study aimed to use the Setaria italica (foxtail millet) genome sequence in the identification of a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring water logging tolerance that will benefit future expansion of its cultivation area.

Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer waterlogging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome to identify and characterize the ortholog from Setaria italica. Softberry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding.

Conclusion: The study is a helpful example of how to advance in genomics research, including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species by utilizing the available genome sequence.

Citation

Nor Akmar Abdullah, S., Abdullah, A., & Mayes, S. (2021). Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-Based Transcriptional Activation. Current Chinese Science, 1(5), 523-533. https://doi.org/10.2174/2210298101666210709104258

• Journal Article Type: Article
• Acceptance Date: Jun 14, 2021
• Online Publication Date: Jul 8, 2021
• Publication Date: 2021-09
• Deposit Date: Jun 16, 2021
• Publicly Available Date: Jul 9, 2022
• Journal: Current Chinese Science
• Print ISSN: 2210-2981
• Electronic ISSN: 2210-2914
• Publisher: Bentham Science Publishers
• Peer Reviewed: Peer Reviewed
• Volume: 1
• Issue: 5
• Pages: 523-533
• DOI: https://doi.org/10.2174/2210298101666210709104258
• Keywords: General Medicine
• Public URL: https://nottingham-repository.worktribe.com/output/5687520
• Publisher URL: https://www.eurekaselect.com/194658/article
• Additional Information: Review Status: Peer Reviewed; Review Process: Single blind; Screening Status: Checked with iThenticate; Received: 2020-11-14; Revised: 2021-04-29; Accepted: 2021-05-10; Published: 2021-11-25