BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize

Abstract

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%–10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs.

Multi-Knock—a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants

“…In the past few years, studies have demonstrated the feasibility of CRISPR-based knockout collections in rice (Oryza sativa L.), maize (Zea mays) and tomato (Solanum lycopersicum) 12,[15][16][17][18][19][20][21][22] . However, thus far, CRISPR/Cas9 has not been used on a genome-scale level to simultaneously target multiple potentially redundant genes in plants or other eukaryotes.…”

Genome editing in maize: Toward improving complex traits in a global crop

“…MGE in maize can also be performed with a larger number of sgRNAs. For instance, vectors harboring up to twelve individual sgRNA expression cassettes have been successfully used for the transformation of an “editor” (i.e., Cas9-expressing) maize line ( Lorenzo et al, 2022 ). Gong et al (2021 ) compared the CRISPR/Cas12a versus the CRISPR/Cas9 system for MGE targeting the maize bZIP transcription factor Opaque2 ( O2 ).…”

“…In BREEDIT, populations transformed with different sets of sgRNAs (twelve different sgRNAs on each set) are then crossed, stacking up mutations in an increasing number of genes. This approach is useful for both trait improvement as well as for the discovery of new genes contributing to a given trait of interest ( Lorenzo et al, 2022 ).…”

Focus on climate change and plant abiotic stress biology

“…Six primary research articles in this issue address topics related to abiotic stress and climate change. Lorenzo et al (2023) present a gene discovery pipeline in maize called BREEDIT, combining multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. Two articles report on proteins that function in guard cells, one related to abscisic acid (ABA)-activated Ca 2+ signaling ( Tan et al, 2023 ) and the second describing a module that regulates microtubule disassembly ( Wang et al, 2023 ).…”