Harnessing Genome Editing Techniques to Engineer Disease Resistance in Plants

Abstract

Modern genome editing (GE) techniques, which include clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and LAGLIDADG homing endonucleases (meganucleases), have so far been used for engineering disease resistance in crops. The use of GE technologies has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, knockdowns, modifications, and the repression and activation of target genes. CRISPR/Cas9 supersedes all other GE techniques including TALENs and ZFNs for editing genes owing to its unprecedented efficiency, relative simplicity and low risk of off-target effects. Broad-spectrum disease resistance has been engineered in crops by GE of either specific host-susceptibility genes ( S gene approach), or cleaving DNA of phytopathogens (bacteria, virus or fungi) to inhibit their proliferation. This review focuses on different GE techniques that can potentially be used to boost molecular immunity and resistance against different phytopathogens in crops, ultimately leading to the development of promising disease-resistant crop varieties.

CRISPR/Cas9-mediated targeted mutagenesis of TAS4 and MYBA7 loci in grapevine rootstock 101-14

“…Genome editing technologies, on the other hand, can result in non-''genetically modified organisms'' (GMO) after outcrossing the effector transgene locus. Recently the USDA issued a directive that the agency does not have plans to regulate plants generated using gene editing techniques that create deletions/insertions that could otherwise have been developed through traditional breeding techniques (https://www.usda.gov/media/ press-releases/2018/03/28/secretary-perdue-issues-usdastatement-plant-breeding-innovation), expanding prospects for genome editing of crops for resistance to insect pests and pathogens (Bisht et al 2019;Mushtaq et al 2019).…”

Modern CRISPR/Cas9 biotechnology is a current challenge to create elite disease resistant crops cultivars

“…As is known, Cas9 has been successfully used to edit the genomes of a number of plants, including barley (Khlestkina, Shumny, 2016;Gerasimova et al, 2017;Mushtaq et al, 2019).…”

“…It is a new popular technology of gene editing in humans, animals and higher plants. The CRISPR/ Cas9 system is a simple, inexpensive and versatile tool for genome editing, resulting in a groundswell of research based on the technique, of which the popularity over the past 6 years has become known as the 'CRISPR craze' (Feng et al, 2013;Brooks et al, 2014;Doudna, Charpentier, 2014;Svitashev et al, 2015;Chandrasekaran et al, 2016;Khlestkina, Shumny, 2016;Gerasimova et al, 2017;Mushtaq et al, 2019). Unlike its predecessors, ZFNs or TALENs, the CRISPR/Cas9 system does not require any protein engineering steps, making it much more straightforward to test multiple gRNAs for each target gene.…”

Catalytically Enhanced Cas9 through Directed Protein Evolution

“…Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective 4,[11][12][13][14] or of low efficiency such as with type II-C Cas9 15-18 or in non-mammals 19,20 . We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9).We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) with up to 4-fold improvement of in vitro catalytic efficiency, as well as the widely used Streptococcus pyogenes Cas9 (SpyCas9), which showed a 2-fold increase in homology directed repair (HDR)-based gene insertion in human colon cancer cells.Functional CRISPR-Cas9 enzymes are composed of a Cas9 protein, a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).…”

“…Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective 4,[11][12][13][14] or of low efficiency such as with type II-C Cas9 [15][16][17][18] or in non-mammals 19,20 . We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9).…”