Genome-editing, a recent technological advancement in the field of life sciences & is one of the techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility.
Cells have several inherent mechanisms for the repair of double-strand DNA breaks (DSBs). These DNA repair mechanisms have been acknowledged as important approaches for targeted gene modification or editing, by introducing precise breaks in the genome at specific sites. Approaches to modify the genomic DNA and RNA included self-splicing introns, cross-linking agents like psoralen or bleomycin or other chemical reagents coupled with chemical recognition of DNA sequences using polyamides or peptide nucleic acids (PNAs), and homing endonucleases encoded by introns. The current advancements for editing genes include site-specific nucleases, usage of which for genome editing began with the advent of zinc-finger nucleases (ZFNs). The ZFNs were the first truly target specific protein reagents that revolutionized the field of genome manipulation. ZFNs are DNA binding domains and specifically recognize three base pairs at the target site.
Recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) technique based on type II prokaryotic adaptive immune system, that helps bacteria or archaea against the invading phages, provides an excellent alternative to the first generation site-directed nucleases. TALENs and ZFNs were successfully used for gene editing, but CRISPR/Cas provides several advantages in terms of design, specificity, multiplexing, cost, and flexibility over other methods.
Cas Variants and Other Nucleases for Plant Genome Editing
- Cas9 is a DNA specific endonuclease that is found in bacterial species—such as Streptococcus pyogenes, Streptococcus aureus, Streptococcus thermophilus, Francisella novocida, and Brevibacillus laterosporus out of which the Streptococcus pyogenes Cas9 (SpCas9) is predominantly used.
- Cas9 is a multifunctional protein having two nuclease domains, HNH and RuvC-like domain. Cas9 can be modified into a nickase, capable of producing a single strand cleavage, by mutating either the HNH or the RuvC-like domain
- More recently, a new class II system encoding a miniature(529 amino acids) effector, Cas14a1, has been identified Importantly, this Cas variant functions as a PAM-independent single stranded DNA nuclease. Many more Cas variants and orthologs are being discovered and exploited for gene editing purposes since the CRISPR/Cas system is a general immune system present in bacteria and archaea for protection against bacteriophages.
Genes Targeted for Genome Editing in Plants
Genome editing has also been used in plants for functional annotation of various genes previously deciphered and proved to be associated with many vital processes.
For example, stress-related genes, ideal marker genes, genes related to plant architecture have been targeted, which are discussed in the subsequent sections.
Gene editing has also been successfully used to improve plant resistance against fungal and bacterial pathogens. Significant efforts have been made towards powdery mildew resistance in several crop species. The use of fungicides can efficiently control powdery mildew, but the rapid evolution of fungal strains to develop resistance to these fungicides and the additional costs to growers, together with the hazardous effect of fungicides on the environment necessitates the development of alternative strategies. The most common practice in developing resistant varieties is targeting susceptible genes (S gene), MLO, which suppress the defence system of plants against powdery mildew. This denotes that loss-of-function mutations in the MLO alleles should lead to broad-spectrum resistance to the powdery mildew. Powdery mildew in wheat is caused by Blumeria graminis f. sp. tritici (Bgt), one of the most damaging plant pathogens in wheat production. have successfully knocked out all of the homologs of the MLO gene in hexaploid wheat by deploying gene editing approach, which renders durable resistance to Bgt in wheat plants In another study, CRISPR/Cas9 edited tomato plants named as “tomelo” were developed to confer resistance against powdery mildew by targeting MLO genes. Similarly, the susceptibility (S) gene, MLO7 has also been targeted in grape for controlling Erysiphe necator infection, a fungal agent that causes powdery mildew in grapes. Here, ribonucleoproteins (RNPs) were used to directly deliver CRISPR/Cas9 reagents to the protoplasts of grape cultivar Chardonnay. A similar approach was used in the same study, for developing apple plants resistant to fire blight pathogen, Erwinia amylovora, which is an enterobacterial phytopathogen. For this purpose, DIPM-1, DIPM-2, and DIPM-4 genes were targeted for genome editing. CRISPR/Cas9 system for genome editing has also been successfully exploited in developing resistance to blast disease in japonica rice by targeting codons close to translation initiation codon of OsERF922 with a sgRNA to introduce indels. These mutant lines further characterized for many agronomic traits including flag leaf width, flag leaf length, plant height, number of panicles, rate of seed setting, length of panicle, and seed weight, and none of the observed traits significantly differed from wild-type plants, implying that alteration of OsERF922 can yield plants with increased resistance without any negative effect on plant development. Genome editing approaches seem promising for combating devastating diseases in crop plants.
Genes for the Enhancement of Abiotic Stress Tolerance in Plants
- Abiotic stresses are the most serious constraints in agricultural production, and the negative impact is bound to worsen with global climate change.
- Abiotic stress tolerance is governed by a number of genes and largely influenced by environmental factor which make it challenging to study.
- Conventional breeding techniques and transgene-based systems, although have helped to develop resilient crop varieties, but the complex inheritance of abiotic stress-related traits and higher environment effects make it very difficult to develop novel cultivars using conventional methods.
- Similarly, induced mutagenesis is a widely explored method for the genetic improvement of several crop species besides being entirely dependent on random events.
- CRISPR/Cas9 system can be utilized for forward genetics where manipulation of genes and gene expression can be performed to study the genetics of abiotic stress response, and thus assist in producing the stress-resistant crop varieties.
- The CRISPR/Cas9 approach, which is now being largely exploited in plant science, is restricted to a very few publications related to its application for the understanding and development of abiotic stress-resistant plants.
CRISPR/Cas based genome editing approach which has improved yield under drought stress
The study has targeted ARGOS8 that negatively regulates ethylene responses. Improved expression of ARGOS8 in genome-edited plants showed enhanced drought tolerance. In another study, a tissue-specific AtEF1 promoter was used to drive truncated gRNAs (tru-gRNAs) and Cas9, which caused mutations in abiotic stress-responsive genes, namely OST2/AHA1, leading to enhanced stomatal responses in Arabidopsis. Rice genes OsRR22 and OsNAC041 have also been targeted to increase salinity tolerance. A recent study has been successful in targeting 25 different genomic targets by leveraging RNase/DNase property of Acidaminococcus Cas12a (Cpf1) for multiplexed genome editing. The approach mentioned above can be helpful for simultaneously targeting the multiple genes involved in abiotic stress.
Aquaporins are some of the prime candidates for abiotic stress enhancement, where genome editing can be employed to modulate solute transport regulations, particularly water, urea, H2O2, and silicon. Similarly, other transporter proteins are also prominent candidates for genome editing for the enhancement of abiotic stress tolerance. These factors indicate that the CRISPR/Cas system can be harnessed prolifically for this novel purpose and will be the future of targeting minor genes of complex quantitative traits related to abiotic stresses.
Editing Polyploidy Genomes—Challenges and Perspective
The introduction of desirable traits in leading crop varieties using classical breeding approaches is a very challenging and time-consuming task when it comes to very complex polyploid genomes such as sugarcane, cotton, wheat, and potato. Introgression of multiple traits and modification of metabolic pathways is also tricky with conventional breeding approaches in polyploidy plants. However, genome editing techniques over several advantages over the conventional breeding process, where multiple genes or metabolic pathways can be targeted at the same time, without any linkage drag. CRISPR/Cas9 can especially be very efficiently used for many purposes and has already been exploited to generate broad-spectrum resistance to powdery mildew in wheat, and has also been used to generate mutations in cotton Duncan grapefruit and potato. CRISPR-Cas will be the most widely used tool for molecular improvement of polyploids in the near future.
Multi-Targeting Genome Editing Approaches
- One of the major and prevalent advantages of CRISPR/Cas9 technology is that it can be used to target multiple genes (or multiple sites within a gene) to create small or large deletions in the genome and provides practical applications in basic and applied biological research.
- In general, two approaches have been used for expressing multiple gRNA. In the first approach, each gRNA is expressed with an individual promoter and in second approach multiple gRNAs expressed by one promoter as a single transcript which is further processed or cleaved to release individual gRNAs.
- Currently, there are several efficient strategies developed to achieve CRISPR/Cas9 enabled multiplex genome editing, which are discussed below.
- t-RNA Mediated Multi-Targeting Genome Editing
- Engineering Introns to Express sgRNAs
- Csy4 Nuclease mediated multi targeting genome editing
- Drosha-Based Multi-Targeting Genome Editing
- Precision Editing/Base-Editing Approach
- t-RNA Mediated Multi-Targeting Genome Editing
- Transfer-RNAs (tRNAs) are a fundamental cellular component of all organisms, and their production and processing are mediated by RNA-processing systems.
- With this concept, Xie et al. (2015) developed an endogenous RNA-processing system to produce multiple gRNA from a single transcript (Figure 3).
- They have shown that a synthesized DNA fragment having tRNA–gRNA in a tandemly arrayed fashion can be proficiently processed into gRNAs having the desired 50 targeting sequences, which precisely directed Cas9 protein for editing multiple chromosomal targets.
- The tRNA-processing system that includes RNaseZ and RNaseP, inherently present in a cell, precisely cleaves 50 and 30 ends of the tRNAs, thereby releasing individual gRNAs.
Engineering Introns to Express sgRNAs
Pol III and Pol II promoters express small nucleolar RNAs (snoRNAs) and microRNAs (miRNAs) respectively. The snoRNA and miRNA are mostly present in the introns of coding genes and processed by their biogenic pathway. Similarly, gRNAs can also be engineered from introns of Cas9 or Cpf1 by modifying the RNA processing machinery to precisely cut individual gRNAs without disrupting the standard splicing mechanism. Since introns are universal modules of a eukaryotic genome, they can be engineered to express gRNAs in virtually all the eukaryotes (Figure 4). Intron PTGs constructs (inPTGs) have comparable fragment deletion frequencies up to 30.9% with the PTG constructs. Full length and truncated introns have also been tested, and not much difference was found in the editing efficiency using these two, revealing that intron length has no profound influence in this method
Csy4 Nuclease mediated multi targeting genome editing
The Csy4 endonuclease , from Pseudomonas aeruginosa has been effectively exploited to excise multiple gRNAs from synthetic polycistronic transcript. Multiple gene expression was observed by designing gRNAs in a tandem array, each flanked by recognition sequence for Csy4. Once bound to the RNA stem-loop, Csy4 cleaves after the guanine at position 20, allowing to generate multiple RNA transcripts. The RNA processing ability of Csy4 can be applied for gene deletion and interference lucratively. ThePTGand cys4 mediated multiplexing has also been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), barley (Hordeum vulgare), wheat (Triticum aestivum), and Medicago truncatula. (Figure 5)
Drosha-Based Multi-Targeting Genome Editing
Drosha based multi-targeting genome editing approach is a multi-target genome editing approach in which tandem consecutively arranged miRNA (or shRNAs)-sgRNA genes are expressed under the control of a single polymerase II promoter. Generally, Pol III promoters are manipulated to express sgRNAs because of the lack of special structures such as 5’cap, 3’ tail or introns, but they are ine_cient because of short length and limited life of Pol III transcripts (Figure 6). Polymerase II transcribed sgRNAs are desired because of their ability to be expressed in a tissue-specific and flexible manner, but these have redundant nuclease activity because of the 50 cap structure. This issue can be rectified by using miRNA-based strategy, using the microprocessor protein complex, comprised of Drosha, an RNase III enzyme, and its cofactor, DGCR8 or Pasha for the production of mature gRNAs and miRNAs. Even though a highly robust approach, it is still relatively less preferred by the plant scientists.
Precision Editing/Base-Editing Approach
Use of CRISPR/Cas approach is straight forward when it comes to generating knockouts, but precise base editing remains challenging because of the preference of NHEJ instead of HDR pathway for the repair of DSB in natural systems. Moreover, HDR also requires an oligonucleotide template (donor template) to be developed and transferred along-with CRISPR/Cas reagents into the cells, for target-specific recombination and gene repair. Methods have been developed to precisely edit bases in DNA without causing DSBs using CRISPR/Cas mechanism by using modified chimeric Cas protein, having a DNA recognition module attached to a catalytic domain with the ability of chemically modifying the bases. The dCas9 guided by a sgRNA is used most of the times in such a system, with reduced insertions and deletions (Figure 7).
CRISPR/Cas based genome editing emerged as a game-changer in recent years due to its enormous potential to make desired modifications in the genome and also for versatile diagnostic purposes. The technological advances are being implemented not only for generating knockouts but also knock-ins, and for activation as well as repression of gene expression. Because of its utility in a practical system, many advancements have already been achieved in a very short period after its discovery, that includes DNA free genome editing systems (RNPs), multiple Cas9 variants, many multi-gene targeting approaches, precise base editing, and measures to increase the frequency of HDR. Advancements in the CRISPR/Cas related bioinformatics tools have also led to the elevated use of this powerful genome editing technique. The CRISPR/Cas based genome editing is still limited to sophisticated molecular biology lab which mostly focuses on the fundamental biological question. In contrast, crop breeders still need to make significant efforts to implement technological advances in crop improvement programs. Pay-per-use basis, public as well as private facilities providing service for construct development, transformation, and evaluation of genome-edited plants, will be game-changer to take the advancements from lab to field by facilitating crop breeder with most efficient genome editing tools. The information provided here will be helpful to understand the recent technological advances, knowledge gaps, problems with technological adaptations which are required for the efficient utilization of genome editing tools for crop improvement.