Combining multiplex gene editing and doubled haploid technology in maize

Area of Science:

• Plant genetics and molecular biology
• Agricultural biotechnology
• Gene editing technologies

Background:

• Multiplex CRISPR/Cas9 gene editing allows simultaneous targeting of multiple genes, but obtaining homozygous mutants for functional analysis is labor-intensive.
• Primary transformants often exhibit hetero-allelic mutations or genetic mosaicism, necessitating extensive breeding and genotyping.

Purpose of the Study:

• To develop a rapid and efficient strategy for generating genetically identical plant lines with various combinations of homozygous edits.
• To facilitate the study of gene families and identify beneficial allele combinations for crop improvement.

Main Methods:

• Combined highly multiplex CRISPR/Cas9 gene editing in Zea mays (maize) with in vivo haploid induction.
• Utilized efficient in vitro generation of doubled haploid plants through embryo rescue doubling.
• Employed three CRISPR/Cas9 constructs targeting 36 genes involved in leaf growth.

Main Results:

• Generated an array of homozygous mutant lines with diverse edit combinations within three generations.
• Identified several genotypes, including a septuple mutant, exhibiting a reproducible 10% increase in leaf size.
• Demonstrated the efficiency of the combined gene editing and doubled haploid strategy.

Conclusions:

• The developed strategy significantly accelerates the production of higher-order homozygous mutants for functional genomics in plants.
• This approach is valuable for studying gene families and discovering allele combinations that enhance quantitative crop traits.
• Facilitates high-throughput phenotyping and accelerates breeding programs for improved crop varieties.