Increasing Fruit Weight by Editing a Cis-Regulatory Element in Tomato KLUH Promoter Using CRISPR/Cas9

Qiang Li; Qian Feng; Ashley Snouffer; Biyao Zhang; Gustavo Rubén Rodríguez; Esther van der Knaap

doi:10.3389/fpls.2022.879642

Increasing Fruit Weight by Editing a Cis-Regulatory Element in Tomato KLUH Promoter Using CRISPR/Cas9

Front Plant Sci. 2022 Apr 11:13:879642. doi: 10.3389/fpls.2022.879642. eCollection 2022.

Authors

Qiang Li^{1

2}, Qian Feng², Ashley Snouffer², Biyao Zhang², Gustavo Rubén Rodríguez^{2

3}, Esther van der Knaap^{2

4

5}

Affiliations

¹ College of Horticulture, Hebei Agricultural University, State Key Laboratory of North China Crop Improvement and Regulation, Key Laboratory of Vegetable Germplasm Innovation and Utilization of Hebei, Collaborative Innovation Center of Vegetable Industry in Hebei, Baoding, China.
² Center for Applied Genetic Technologies, University of Georgia, Athens, GA, United States.
³ Instituto de Investigaciones en Ciencias Agrarias de Rosario (IICAR-CONICET-UNR), Cátedra de Genética, Facultad de Ciencias Agrarias UNR, Santa Fe, Argentina.
⁴ Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA, United States.
⁵ Department of Horticulture, University of Georgia, Athens, GA, United States.

Abstract

CRISPR/Cas-mediated genome editing is a powerful approach to accelerate yield enhancement to feed growing populations. Most applications focus on "negative regulators" by targeting coding regions and promoters to create nulls or weak loss-of-function alleles. However, many agriculturally important traits are conferred by gain-of-function alleles. Therefore, creating gain-of-function alleles for "positive regulators" by CRISPR will be of great value for crop improvement. CYP78A family members are the positive regulators of organ weight and size in crops. In this study, we engineered allelic variation by editing tomato KLUH promoter around a single-nucleotide polymorphism (SNP) that is highly associated with fruit weight. The SNP was located in a conserved putative cis-regulatory element (CRE) as detected by the homology-based prediction and the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Twenty-one mutant alleles with various insertion and deletion sizes were generated in the LA1589 background. Five mutant alleles (m2_+4bp , m3_+1bp , m5_-1bp , m13_-8bp , and m14_-9bp ) showed a consistent increase in fruit weight and a significant decrease in the proportion of small fruits in all experimental evaluations. Notably, m2_+4bp and m3_+1bp homozygote significantly increase fruit weight by 10.7-15.7 and 8.7-16.3%, respectively. Further analysis of fruit weight based on fruit position on the inflorescence indicated that the five beneficial alleles increase the weight of all fruits along inflorescence. We also found that allele types and transcriptional changes of SlKLUH were poor predictors of the changes in fruit weight. This study not only provides a way of identifying conserved CRE but also highlights enormous potential for CRISPR/Cas-mediated cis-engineering of CYP78A members in yield improvement.

Keywords: CRISPR/Cas; KLUH; cis-regulatory element; fruit weight; promoter; tomato.