Crop Improvement Using CRISPR/Cas9 Systems: Advances and Challenges

Saumya Shah
CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, Uttar Pradesh, India
Correspondence to: saumyashah600@gmail.com

DOI: https://doi.org/10.70389/PJPB.100004

Additional information

• Ethical approval: N/a
• Consent: N/a
• Funding: No industry funding
• Conflicts of interest: N/a
• Author contribution: Saumya Shah – Conceptualization, Writing – original draft, review and editing
• Guarantor: Saumya Shah
• Provenance and peer-review:
  Commissioned and externally peer-reviewed
• Data availability statement: N/a

Keywords: Abiotic stress tolerance, Cas9, Crispr, Crop improvement, Disease resistance, Genome editing.

Peer Review
Received: 1 September 2024
Revised: 7 November 2024
Accepted: 10 November 2024
Published: 21 November 2024

Introduction

With the increasing population of the world, agriculture is facing great pressure to meet the global demand for food, feed, fiber, and bioenergy. The global climate change is causing extreme weather changes, leading to heat and drought stress: as a result, farmers around the world are facing significant losses in crop yield.¹ Crop improvement is an important aspect of agricultural research that aims to increase crop productivity, resilience, and sustainability (https://www.era-ard.org/crop-improvement/). There are a variety of approaches to crop improvement, which include conventional breeding, biotechnology, and genetic engineering.² Conventional crop improvement techniques involve hybridization, selection, and breeding.³ This crop improvement practice dates back centuries and involves crossbreeding crops to achieve desirable traits, such as disease resistance, drought resistance, and higher yields. However, it can be time-consuming and not always produce the desired results.² Biotechnology and genetic engineering have transformed crop improvement by involving the transfer of genes of desired traits via an exogenous T-DNA cassette into different organisms.⁴ This process, also known as gene manipulation, has led to the development of genetically modified organisms (GMOs) with better quality traits, such as pest resistance, high yield, and nutritional values. Despite their potential benefits, the research and development of GMOs are still costly and time-consuming. Also, ­concerns about safety regulations, environmental impact, and social acceptance demands persist.⁵

Recent advances in biotechnology offer us more clear information and insights into the biochemical and molecular mechanisms used to edit a genome and alter downstream pathways. Genome editing (GE) technology is a precise and predictable method of editing a plant’s genome, which has shown significant benefits in crop improvement.⁶ Sequence-specific nucleases such as zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the most advanced clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein ⁹ (Cas9) are some cutting-edge techniques which modify the plant genome more precisely with a low probability of generating off-targets.⁷ These GE technologies use programmable nucleases for the generation of double-stranded DNA breaks (DSBs) to increase the specificity of the target locus.³ ZFNs are chimerically engineered targeted DNA cleavage proteins that cut DNA sequences at specific sites. They facilitate targeted GE by generating DSBs in the DNA to replace the gene by homologous recombination (HR). Each ZFN consists of a DNA-binding domain with a chain of two-finger modules that recognizes a unique 6-bp hexamer in the DNA sequence and a DNA-cleaving domain consisting of a FokI nuclease domain (Figure 1A).

Dimerization of the FokI domain is critical for ZFN enzymatic activity (Figure 1B). These domains join to form a zinc finger protein. When fused, these domains form a highly specific genomic scissor. ZFN-mediated gene editing manipulates the genome of any organism permanently by introducing site-specific DSBs into the DNA sequence. This DSB in the DNA sequence is repaired by either non-homologous end joining (NHEJ) or HR to produce the desired mutation or recombination of the gene.⁸ TALENs, another efficient GE tool, is an alternative to ZFNs (Figure 2A). TALENs also comprise a non-specific FokI endonuclease (Figure 2B) similar to ZFNs and specific DNA-binding domains of highly conserved repeats derived from transcription activator-like effectors (TALEs). TALE proteins are produced by pathogenic Xanthomonas bacteria that secrete TALEs to alter gene transcription in host plants.⁹ The CRISPR/Cas9 system is the most preferred method over ZFNs and TALENs. This technique is simple, accurate, efficient, and cost-effective, which has attracted the attention of GE communities. This was considered the biggest advancement in the scientific world, which has opened a new avenue for sustainable improvement in agriculture.¹⁰

This review focuses on the recent advancements and challenges of CRISPR/Cas systems. We have also ­summarized the application and prospects of CRISPR/Cas systems in crop improvement.

A Brief Overview of the CRISPR/Cas9 System

CRISPR are a class of spacer DNA sequences found in the genome of prokaryotes, such as bacteria and archaea. These CRISPRs are derived from viral DNA fragments that had previously infected the prokaryotes.¹¹ The CRISPR-Cas systems act as a defense mechanism that detects and destroys the DNA from similar viruses to prevent repeated infections. CRISPR-Cas systems are grouped into two different classes. Class 1 systems include a complex of multiple Cas proteins that degrades foreign DNA. Conversely, Class 2 systems utilize a single large Cas protein to degrade foreign DNA. Class 1 is further grouped as types I, III, and IV, while class 2 is classified into types II, V, and VI.¹²

These 6 system types are further categorized into 33 subtypes. Each type and most subtypes are distinguished by a “signature gene” that occurs almost ­exclusively in the category.¹³ Commonly, CRISPR-Cas systems have a Cas1 protein. The GE tool CRISPR/Cas9 is an RNA-guided acquired immune system that has been exploited from Streptococcus pyogenes.7 The mechanism of CRISPR/Cas9-mediated immunity in S. pyogenes can be divided into three different steps: The first stage is adaptation or spacer acquisition: In this phase, a protospacer from the viral DNA is incorporated into the CRISPR locus, generating a new spacer. Generally, Cas1 and Cas2 proteins are widely involved in the spacer acquisition process as these proteins are present in almost all CRISPR-Cas types.¹⁴ The second stage is the expression stage in which the biogenesis of CRISPR RNA (crRNA) occurs. This technology aids in immunization by expressing Cas genes and transcribing the CRISPR array into a long precursor crRNA (pre-crRNA). Cas proteins and the accessory factors further process the pre-crRNA into a mature guide crRNA. This mature crRNA has memorized sequences of the invading virus.15 The third stage is interference where the mature crRNA and Cas proteins work together to identify and degrade the target nucleic acid.14,15 The detailed representation of CRISPR/Cas9-mediated immunity in S. pyogenes is shown in Figure 3.

Type II CRISPR system is used for GE with the CRISPR-Cas9 system. This system includes:

• Ribonucleoprotein: It uses a Cas9 enzyme. The active form of this enzyme has the ability to alter DNA. Owing to its ability to recognize DNA, the Cas enzyme exists in a wide variety (for example cas9, cas12, cas13 etc) that have different functions, such as single-strand nick­ing, double-strand breaking, and DNA binding.
• crRNA: It contains a guide RNA which locates the correct segment of the host DNA along a region that binds to trans-activating CRISPR RNA (tracrRNA) to form an active complex, forming a hairpin loop.
• tracrRNA: It binds to crRNA and forms an active complex.
• sgRNA: It is a single-guide RNA that is a combined RNA consisting of a tracrRNA and at least one crRNA.
• DNA repair template: It is a DNA molecule that is used by the host cell as a template for the DNA repair process, allowing insertion of a specific DNA sequence into the host segment broken by Cas9. This occurs by the generation of the DSB and the subsequent DNA repair. Thereafter, a cell repair is initiated through a process known as an NHEJ. This process is error-prone and hence results in mutations by involving small insertions and deletions (indels) in target sites. Therefore, the target genomic elements such as regulatory regions and gene functions are eliminated or interrupted. The second repair process that can also be initiated is the homology direct repair-mediated gene replacement. It is an error-free process that facilitates precise editing of genes, DNA or regulatory elements ( Figure 4).¹⁶

In 1987, Ishino et al. at Osaka University discovered CRISPRs in Escherichia coli.¹⁷ However, the role of CRIS­PRs in bacterial immunity remained unidentified until Mojica et al. identified short regularly spaced repeats (SRSPs) in 20 species of microbes as belonging to the same family.¹⁸ In 2012, a seminal study was published by Jennifer Doudna and Emmanuelle Charpentier that demonstrated CRISPR-Cas9 as a gene- editing tool.19 For this robust genome-editing tool, they received The Nobel Prize in Chemistry. The timeline of key discoveries and advances is shown in Table 1.

Table 1: The Timeline of Key Discoveries and Advances in CRISPR/Cas9.
S. No.	Key Discoveries and Advances	Year	Reference
1.	CRISPRs were first discovered by Ishino et al. at Osaka University in Escherichia coli.	1987	17
2.	A group of Dutch scientists reported a study on a cluster of interrupted direct repeats (DRs)  in Mycobacterium tuberculosis.	1993	20
3.	Mojica et al. identified short regularly spaced repeats (SRSRs) in 20 species of microbes as belonging to the same family.	2000	18
4.	Mojica and Ruud Jansen proposed the term CRISPR (clustered regularly interspaced short palindromic repeats).	2001	21
5.	A report showed CRISPR repeat regions from the genome of Archaeoglobus fulgidus.	2002	22
6.	According to Rodolphe Barrangou, Streptococcus thermophilus acquires increased phage resistance due to the integration of additional CRISPR spacer sequences after recurrent phage infections.	2005	23
7.	Three different research studies revealed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA, such as plasmids.	2005	24–26
8.	The first report published in 2007 showed evidence that CRISPR functions as an adaptive immune  system.	2007	27,28
9.	A complex of Cas protein known as Cascade was identified by Brouns and Van der Oost in E. coli.	2008	29
10.	A study published in 2010 showed that CRISPR-Cas cleaves strand phage and plasmid DNA in S. thermophilus.	2010	30
11.	Jennifer A. Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease as a two- component system by combining the two RNA molecules into a single-guide RNA so that when paired with Cas9, it could locate and cleave the  target DNA indicated by the guide RNA.	2012	19
12.	A US patent with number 8,697,359 was awarded to Feng Zhang and nine other members of the Broad Institute of MIT and Harvard.	2014	31
13.	CRISPR was first applied to tomatoes.	2014	32
14.	Chinese scientists P. Liang and Y. Xu published the first report showing that the CRISPR-Cas9 system can efficiently make gene edits in human tripronuclear zygotes.	2015	33
15.	Emmanuelle Charpentier and Jennifer A. Doudna were jointly awarded The Nobel Prize in Chemistry for their contribution in developing a robust method for genome editing.	2020	34
16.	The first small clinical trial of intravenous CRISPR gene editing in humans with promising results was  published in 2021.	2021	35
17.	The first CRISPR-edited food was accepted  publicly for sale in Japan.	2021	36
18.	The first CRISPR gene-edited marine animal/seafood was  reported.	2021	37
19.	Medicines and Healthcare Products Regulatory Agency (MHRA) of the United Kingdom approved Casgevy, the first drug based on CRISPR gene editing, to treat sickle cell anemia and beta-thalassemia.	2023	38
20	The FDA approved the first gene therapy in the US for the treatment of patients with sickle cell disease  (SCD).	2023	39

Recent Advances in CRISPR/Cas9 in Crop Improvement

GE using CRISPR/Cas9 creates predictable and inheritable changes in specific sites of a genome. It has a low probability of off-targeting and no integration of exogenous gene sequences. Hence, when applied for crop improvement, GE using CRISPR/Cas9 can be a cost-effective process for achieving desired traits, developing resistance in crops, and biofortification.6,⁴⁰ There are several examples published recently which show the potential application of CRISPR/Cas9 in crop improvement.⁴¹

External traits (such as size, color, texture, and fragrance) and internal traits (such as proteins, starch, lipids, carotenoids, lycopene, γ-aminobutyric acid [GABA], and flavonoids) significantly influence the commercial value of crops.6 Achary and Reddy^20,21 demonstrated that CRISPR/Cas9-mediated alterations in Grain Width and Weight2 (GW2) locus improve the aleurone layer and grain nutritional quality in rice.⁴² Similarly, Usman et al.⁴³ used CRISPR/Cas9 technology-generated GS3 mutants that exhibited increased grain size without any change in other agronomic traits. The CRISPR/Cas9 system was also applied to generate pink-fruited tomato plants by knocking out SlMYB12 genes in four elite red-fruited inbred lines.⁴⁴ In another study, it was revealed that CRISPR/Cas9-mediated alterations in the phytoene synthase (PSY1) gene resulted in yellow-colored tomatoes.⁴⁵ Yu et al.⁴⁶ generated tomato lines with long shelf lives using CRISPR/Cas9-induced targeted mutagenesis and gene replacement. The CRISPR/Cas9 technology was also used to introduce aroma into an elite non-aromatic rice variety ASD16 by producing novel ­alleles of OsBADH2.⁴⁷

Yinong Yang, a plant pathologist at Pennsylvania State University, used CRISPR/Cas9 to knock out the polyphenol oxidase (PPO) gene in Agaricus bisporus to achieve anti-browning properties.⁴⁸ Johnston, an Iowa-based DuPont Pioneer and his team, used this technique to produce high-amylopectin corn.⁴⁸ Targeted gene insertion of marker-free DNA in rice using CRISPR/Cas9 resulted in carotenoid-enriched rice.⁴⁹ Recently, researchers have developed GABA-fortified rice by CRISPR/Cas9-induced targeted deletion of the calmodulin-binding domain from rice glutamate decarboxylase 3 (OsGAD3). The modified rice resulted in a seven-fold increased GABA content.⁵⁰ A study showed that CRISPR/Cas9 can be used to develop a low-gluten, non-transgenic wheat. In this study, scientists generated durum wheat lines with lower levels of α-gliadins in the seed kernel.⁵¹ Researchers also edited all the copies of BnaFAD2 using CRISPR/Cas9 technology to generate novel allelic variations in fatty acid levels. In this study, it was revealed that the mutated lines had an 80% increased oleic acid content than the wild type of 66.43%.⁵²

This technology has also been greatly exploited for the development of enhanced disease-resistant plants. A study published in 2020 showed that targeted CRISPR/Cas9-mediated mutagenesis of grapevine mildew resistance Locus O (MLO) genes (VvMLO3 and VvMLO4) resulted in VvMLO3-edited grapevine lines with improved resistance toward powdery mildew.⁵³ Similar to this, the CRISPR/Cas9 technology was employed in wheat to produce powdery mildew-resistant Taedr1 mutant lines.⁵⁴ The promoter region of the OsSWEET14 gene was engineered using the CRISPR-Cas9 technology to develop resistance against bacterial blight.⁵⁵ Another study showed that tomatoes can develop resistance against Tomato yellow leaf curl virus (TYLCV) by using CRISPR-Cas9-mediated targeted editing of the TYLCV genome. This was achieved by generating sgRNA-expressing lines that targeted either the CP sequence or the Rep sequence of the TYLCV genome. However, the sgRNA-expressing lines that targeted the CP sequence of TYLCV showed better viral interference.⁵⁶

CRISPR-mediated GE could be employed to combat and adapt to abiotic stresses like drought, salinity, water logging, heat, and cold stress by characterizing genes that can withstand various abiotic stresses. Zhou et al.⁵⁷ demonstrated that CRISPR/Cas9-based loss-of-function mutants of root-specific NF-YB transcription factor, PdNF-YB21, enhanced root growth and drought resistance via ABA-mediated auxin transport in Populus. Similarly, CRISPR/Cas9-based PdGNC showed drought resistance by activating PdHXK1 (a hexokinase synthesis key gene) expression in Populus.⁵⁸ CRISPR/Cas9-induced slmapk3 mutants showed more tolerance to heat stress in tomato plants.⁵⁹ Zeng et al.60 reported that CRISPR/Cas9-based editing of OsPIN5b, GS3, and OsMYB30 genes exhibited higher yield and enhanced cold tolerance in rice. Also, CRISPR/Cas9-induced precise deletion of hybrid proline-rich protein 1 (HyPRP1) domains showed higher yield and enhanced cold tolerance in tomatoes.⁶¹

Challenges in Using CRISPR/Cas9

Despite being an efficient and precise system for GE, CRISPR/Cas9 technology comes with its own set of challenges. Agrobacterium-mediated gene transformation technique is a very simple, robust, and commonly used method for gene delivery in plants.⁶² However, the success of this method depends upon the choice of the plasmid and cultivar used.7 Several findings suggested that the Agrobacterium rhizogenes-mediated transformation system may have been responsible for low transformation efficiency in rice,^43,63 soybean,^64–66 and tomato⁶⁷ GE. Culture conditions may also influence the rate of infections and regeneration of agrobacterium-infected explants, affecting the reproducibility and efficiency in many plants like soybean,68 clover,⁶⁹ and cassava.⁷⁰ Although a high degree of success is observed in Arabidopsis thaliana where agrobacterium-mediated transformation occurs using the floral dip method, this is not feasible in other plants. For this reason, regeneration of transgenic plants from explant-derived calluses would be required.⁶² Also, further studies and protocol standardization are required to enhance the transformation efficiency of agrobacterium-mediated delivery systems in diverse plant species.

In addition to the delivery system, many other factors may significantly impact the efficiency of CRISPR/Cas9-mediated GE, for example, the position of genes on chromatin. The gene location at the euchromatin region is more effective than that at heterochromatin.⁷¹ Also, the form of Cas9 (i.e., DNA, mRNA, or protein),⁷² the number of sgRNAs used for single-gene knockout editing,73 the length of sgRNAs,74 and the Cas9/sgRNA threshold expression level also tend to impact the efficiency of the delivery system.⁷⁵ CRISPR/Cas9-mediated GE has wide applications in plants. However, there exist certain limitations for GE of polyploid plants.⁷⁶ Due to functional redundancy between para-homologous and homologous genes, it is necessary to delete all copies of genes with the same function at the same time, in polyploid plants. Optimization of the Cas9 codon, promoter, and GC content of the target sequence may influence the mutation effectiveness of polyploid crops.⁷⁷ Furthermore, designing sgRNAs in polyploid plants is also very complicated.

Conclusion

New crop-breeding techniques such as CRISPR/Cas9 is an emerging technology for GE. This technology is providing several promising improvements to crops, from yield enhancements and nutritional quality improvements to strong resistance against disease and abiotic stresses. The successful application includes improved grain qualities, disease-resistant plants, and biofortified crops. All these examples show the potential role of CRISPR/Cas9 in addressing critical agricultural problems. However, still there also exist certain challenges which need to be addressed. The challenges include inefficient agrobacterium-mediated plant transformation and complicated designing of sgRNAs for polyploid crops.

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Cite this article as:
Shah S. Crop Improvement Using CRISPR/Cas9 Systems: Advances and Challenges. Premier Journal of Plant Biology 2024;1:100004

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