CRISPR/Cas editing tools are rapidly advancing precise genome modifications, enabling a broad range of curative applications and diagnostics for human disease. Beyond human health, CRISPR/Cas applications in agriculture are perhaps less well known. Nevertheless, investigators are increasingly harnessing the power of CRISPR/Cas9 for plant gene editing to improve crop efficiency, ensuring sustainable food sources for our ever-expanding global population.
Powdery mildew, the whitish dust that may appear spotted on leaves, shoots, flowers, and even fruits, is a fungal disease that affects a broad range of plant varieties worldwide. With over 650 known species, powdery mildew fungal infections wreak havoc on food supply chains by damaging and reducing production by critical crops, such as barley and wheat.
Broad-spectrum natural resistance to this type of fungal infection was discovered in the 1940s and linked to a loss-of-function mutation in barley’s mildew resistance locus o (mlo). Since then, mlo based resistance has been identified and introduced in many plant varieties for its broad and long-lasting protection (Acevedo-Garcia et al. 2014, Kusch and Panstruga 2017).
“Schematic representation of the complete barley HvMLO protein. The orange bar represents the plant membrane. Colored dots indicate the amino acids of the corresponding mlo-mutants in different plant species.” Retrieved from Yan et al. 2021 (https://creativecommons.org/licenses/by/4.0/). The mlo gene family encodes integral membrane proteins having seven membrane-spanning domains. The exact biochemical function of MLO proteins is not fully known; nevertheless, they are thought to play a role in calcium and calmodulin-dependent signaling.
Plant susceptibility genes (S) genes, such as the mlo gene family, may be edited to induce pathogen resistance. In fact, loss-of-function S gene mutations are well known to protect against a broad range of pathogens. Nevertheless, a common disadvantage to mlo loss-of-function mutations is the resulting negative impact on plant growth and productivity. This caveat has deterred the broad implementation of mlo loss-of-function mutations in agriculture.
Gene editing enabled by CRISPR/Cas9 allows investigators to introduce precise changes in mlo genes to harness the benefits of powdery mildew resistance while retaining optimal plant growth and yield properties. This approach has been leveraged recently by Dr. Caixia Gao’s laboratory at the State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China (Li et al. 2022).
Previously, Dr. Caixia Gao’s group had developed a mutant wheat (Triticum aestivum- Ta) variety missing the expression of all TaMLO1 homologs, which are encoded by A, B, and D genes. Nevertheless, together with strong powdery mildew resistance, the resulting mutant variety, Tamlo-aabbdd, inherited some deficiencies in growth and productivity.
Serendipitously, Gao’s team identified a different Talen-induced wheat mutant that thrived better while conserving strong resistance to powdery mildew. Digging deeper, the team found a large 304 kb deletion which extended into the second exon of the TaMLOB1 gene in the new wheat mlo mutant they named Tamlo-R32.
Interestingly the team determined that the large deletion significantly induced the expression of TaTMT3B, a gene located immediately upstream from the deletion and mostly silenced in wild-type wheat plants. Furthermore, through local chromatin and transcript analysis, Gao’s team identified changes in the chromatin’s conformation, markers (i.e., H3K27me3 and H3K27ac), and transcript density, which enhanced transcriptional activity within the TaTMT3B locus.
Next, by leveraging the specificity of CRISPR/Cas9 based genome editing, Gao’s group removed the TaTMT3B gene from the Tamlo-R32 mutant plants. This approach allowed them to prove the critical role of TaTMT3B for desirable plant properties, as in its absence, the resulting plants consequently showed reduced growth and productivity. Hence, overexpressing TaTMT3B in their triple mutant plants, Tamlo-aabbdd, averted the undesirable phenotypes while retaining resistance to powdery mildew. Ultimately, Gao’s team applied their discoveries and the CRISPR/Cas9 system to develop new wheat varieties harboring the beneficial deletions (i.e., TaMLO-A1, TaMLO-D1, and B genome’s 304 kb deletion).
CRISPR/Cas9 based genome editing is expediting the process of developing new plant varieties, a clear advantage over more time-consuming traditional introgression or backcrossing strategies. While CRISPR/Cas9 gene-editing tools used by Gao’s team included both GenScript-sourced Cas9/sgRNAs plasmids and ribonucleoprotein (RNP) complexes, the use of RNP complexes provided the benefit of eliminating the potential for genomic integration.
Created with BioRender.com
Want to learn how to use CRISPR/Cas9 RNP for gene editing? Download Manual
