Title:Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-Based Transcriptional Activation
Volume: 1
Issue: 5
Author(s): Siti N.A. Abdullah*, Sean Mayes and Mahdi Moradpour
Affiliation:
- Faculty of Agriculture, University Putra Malaysia 43400 UPM, Serdang, Selangor,Malaysia
Keywords:
CRISPR/dCas9, CRISPRa, sgRNA, Setaria italica, water logging tolerance, ERFVII.
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 used 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 the 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 the 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 water logging
tolerance. The study provides a step-by-step example for using publicly accessible databases and
bioinformatics tools from NCBI and Phytozome for the identification and characterization of the
ortholog from Setaria italica. Soft berry 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 useful example of how to advance in genomics research including the
revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species,
through the utilization of the available genome sequence.
