Combining multiplex gene editing and doubled haploid technology in maize

Abstract

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain heteroallelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping.We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling.By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination.We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

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“…Crossbreeding of plants, including natural and artificial hybrids, is an important approach for the generation of new cultivars. In plant breeding, large numbers of genetically stable and homozygous individuals are necessary, and complete homozygote breeding materials are rapidly obtained with doubled haploid technology, which is currently a routine part of breeding programs [39,40]. In this study, according to the 16 pairs of SSR markers, most of the cultivars had homozygous genotypes, which may be conducive to the inheritance of target genetic information in cross-breeding.…”

From Petri Dish to Field: Plant Tissue Culture and Genetic Engineering of Oats for Improved Agricultural Outcomes

“…Furthermore, the principle of haploid induction (either with wide crosses or haploidy inducers), coupled with site-directed mutagenesis, presents a promising strategy for generating mutated plants that are devoid of T-DNA and exhibit a high degree of homozygosity. Such a methodology has the capacity to mitigate the influence of genotype on site-directed mutagenesis outcomes [71,72].…”

Unlocking Nature’s Clock: CRISPR Technology in Flowering Time Engineering

“…In this context, using inventive techniques and approaches to increase efficiency and save time is critical to the success of molecular breeding projects. Recent advances include improvements in delivery using improved Agrobacterium strains [208,209], viral delivery [210], improvements in regeneration using morphogenic regulators [211,212], and new approaches to use multiplex gene editing to study many genes simultaneously [213,214].…”