Developing a pipeline for identification, characterization and molecular editing of cis-regulatory elements: A case study in potato

This study is led by Professor Zixian Zeng (Department of Biological Science, College of Life Sciences, Sichuan Normal University, Chengdu 610101, China). The authors developed a pipeline to identify, characterize and edit this CRE, and further manipulate the expression pattern of gene as well as the resulting phenotypes. The pipeline consists of four main steps, including1) genome-wide prediction of CREs using open chromatin strategy detected by DNase-seq, MNase-seq or ATAC-seq; 2) quick functional validation of candidate CREs using transient transformation with firefly luciferase (LUC) reporter system in tobacco; 3) activity and tissue specificity evaluation of CREs with β-glucuronidase (GUS) reporter system in native plant; 4) genome-editing of CREs, expression evaluation of target genes and phenotyping.

StCDF1 was used as an example. To identify the core promoter region of StCDF1, the authors utilized previously constructed DNase-seq libraries from DM 1-3 leaf tissue and detected a 288 bp DNase I hypersensitive site (DHS) located across upstream and 5’UTR regions of the gene, designated as T#01. In tobacco transient assay and genetics transformation, T#01 is associated with promoter activity, which is mainly distributed in leaf, stem and roots of the potato seedlings. In addition, the promoter T#01 is more active under SD than LD, which is consistent with the expression pattern of StCDF1. To further evaluate the regulatory role of T#01 in vivo, the authors generated a homozygous CRISPR/Cas9-edited line with two deletions (10 bp and 5 bp). The expression patterns of StCDF1 in wild type (WT) and homozygous CRISPR/Cas9-edited line (t#01) showed similar fluctuation between day and night, under both LD and SD conditions. Intriguingly, the CRISPR/Cas9-edited line t#01 displayed significantly lower level of StCDF1 at those time points under both LD and SD conditions. In addition, the deletions in T#01 delayed the tuberization time in both LD and SD conditions with approximately 6 days under LD condition and 5 days under SD condition. The deletions in T#01 also delayed flowering under LD condition and reduced the biomass under SD condition.

Followed this pipeline, the authors identified a 288 bp-core promoter region of StCDF1, which showed photoperiodic inducibility. Genome-editing of the core promoter displayed reduced expression levels and resulting in late tuberization under both long-day and short-day conditions. This pipeline provides an alternative way to improve a specific trait with limited downside on other phenotypes.

See the article:

Developing a pipeline for identification, characterization and molecular editing of cis-regulatory elements: a case study in potato

https://link.springer.com/article/10.1007/s42994-024-00185-1