# Targeting dCas9‐SunTag to a Susceptibility Gene Promoter Is Sufficient for CRISPR Interference

## TL;DR

This paper shows that using CRISPR interference to target susceptibility genes in cassava can reduce disease caused by viruses.

## Contribution

The study demonstrates that CRISPR interference, not DNA methylation, is sufficient to reduce gene expression in cassava brown streak disease.

## Key findings

- Targeting the promoters of nCBP-1 and nCBP-2 with a dCas9-DRMcd-SunTag system reduced gene expression.
- CRISPR interference, rather than DNA methylation, was found to be responsible for the observed gene silencing.
- Future research will test if methylation alone can confer resistance to cassava brown streak disease.

## Abstract

Cassava production in sub‐Saharan Africa is severely impacted by diseases. Most pathogens require interaction with host susceptibility factors to complete their life cycles and cause disease. Targeted DNA methylation is an epigenetic strategy to alter gene expression in plants, and we previously reported that a zinc‐finger fused to DMS3 could establish methylation at the promoter of MeSWEET10a, a bacterial susceptibility gene, and this resulted in decreased disease. Here, we attempt a similar strategy for cassava brown streak disease. This disease is caused by the ipomoviruses CBSV and UCBSV. These viruses belong to the family Potyviridae, which has been shown extensively to require host eIF4E‐family proteins to infect plants and cause disease. We previously found that cassava plants with simultaneous knockout mutations in two eIF4E genes, nCBP‐1 and nCBP‐2, resulted in decreased susceptibility to CBSD. Here, we report successful simultaneous targeting of both promoters with methylation using a dCas9‐DRMcd‐SunTag system. However, in contrast to our previous work with MeSWEET10a, controls indicate that CRISPR interference is occurring in these lines and is sufficient for the reduction of gene expression. Future research will use genetic crosses to segregate away the DNA methylation reagents and, if DNA methylation proves heritable, assess whether methylation alone is sufficient to increase resistance to CBSD.