CRISPR/Cas: A powerful tool for gene function study and crop improvement
Open accessAbstract
It is a long-standing goal of scientists and breeders to precisely control a gene for studying its function as well as improving crop yield, quality, and tolerance to various environmental stresses. The discovery and modification of CRISPR/Cas system, a nature-occurred gene editing tool, opens an era for studying gene function and precision crop breeding. In this review, we first introduce the brief history of CRISPR/Cas discovery followed the mechanism and application of CRISPR/Cas system on gene function study and crop improvement. Currently, CRISPR/Cas genome editing has been becoming a mature cutting-edge biotechnological tool for crop improvement that already used in many different traits in crops, including pathogen resistance, abiotic tolerance, plant development and morphology and even secondary metabolism and fiber development. Finally, we point out the major issues associating with CRISPR/Cas system and the future research directions. Key Scientific Concepts of Review: CRISPR/Cas9 system is a robust and powerful biotechnological tool for targeting an individual DNA and RNA sequence in the genome. It can be used to target a sequence for gene knockin, knockout and replacement as well as monitoring and regulating gene expression at the genome and epigenome levels by binding a specific sequence. Agrobacterium-mediated method is still the major and efficient method for delivering CRISPR/Cas regents into targeted plant cells. However, other delivery methods, such as virus-mediated method, have been developed and enhanced the application potentials of CRISPR/Cas9-based crop improvement. PAM requirement offers the CRISPR/Cas9-targted genetic loci and also limits the application of CRISPR/Cas9. Discovering new Cas proteins and modifying current Cas enzymes play an important role in CRISPR/Cas9-based genome editing. Developing a better CRISPR/Cas9 system, including the delivery system and the methods eliminating off-target effects, and finding key/master genes for controlling crop growth and development is two major directions for CRISPR/Cas9-based crop improvement.
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AAV delivery in vivo may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [101]
The Future perspectives section (‘Off-target effect is a big concern for CRISPR/Cas application’; page unavailable) says delivery method affects off-target editing and that short-lived RNA or protein delivery reduces the chance of cutting other locations. It does not discuss AAV delivery or say that off-target mutations accumulate during in vivo use. (LLM, conf=0.77)
Experimental — accuracy not yet measuredLNP delivery in vivo eliminates off-target effects.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [101]
The “Off-target effect is a big concern” section (page not provided) says directly delivered CRISPR/Cas proteins or RNAs, such as RNPs, can be quickly degraded and significantly reduce off-target effects. It does not establish that LNP delivery in vivo eliminates them; the “RNA and protein-level’s delivery” section likewise says RNP transformation may reduce off-target effects. (LLM, conf=0.87)
Experimental — accuracy not yet measuredDesigning sgRNA is more convenient, programmable and cost-effective than designing DNA binding domains.
“Designing sgRNA is more convenient, programmable and cost-effective than designing DNA binding domains, thus CRISPR/Cas9 is more favored than ZFNs and TALENs and has revolutionized the biotechnology field (Zhang et al., 2021; Tran et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Zhang et al., 2021
CRISPR/Cas9 is more favored than ZFNs and TALENs and has revolutionized the biotechnology field.
“Designing sgRNA is more convenient, programmable and cost-effective than designing DNA binding domains, thus CRISPR/Cas9 is more favored than ZFNs and TALENs and has revolutionized the biotechnology field (Zhang et al., 2021; Tran et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Zhang et al., 2021
AAV delivery in vivo lasts for years in terminally differentiated cell types.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [101]
The CRISPR/Cas delivery methods section discusses plant virus vectors, and Future perspectives discusses persistent expression from T-DNA in plants. Neither section addresses AAV delivery or whether it lasts for years in terminally differentiated cells (searched: AAV and adeno-associated virus with those terms). (LLM, conf=0.76)
Experimental — accuracy not yet measured
LNP delivery in vivo can be quickly degraded.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [101]
The ‘Off-target effect is a big concern for CRISPR/Cas application’ section says delivered Cas proteins and guide RNAs can be quickly degraded in plant cells. It does not discuss LNP delivery or whether LNPs degrade in vivo; searches for LNP and lipid nanoparticles found no passage addressing that claim. No page number was available in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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- Authors
- Zhang, D., Zhang, Z., Unver, T., & Zhang, B.
- Affiliations
- Henan Agricultural University (CN)
- Henan Institute of Science and Technology (CN)
- East Carolina University (US)
- Corresponding author
- Zhang, Dangquan
- Zhang, Zhiyong
- Zhang, Baohong
- Venue
- Journal of Advanced Research
- Publisher
- Elsevier BV
- Published
- 2021-03
- Volume
- 29
- Pages
- 207-221
- ISSN
- 2090-1224 (ISSN-L)
- 2090-1232
- PMC
- PMC8020163
- OpenAlex
- W3094156280
- Cited by
- 373
- References
- 147
- Citations by year
- 2026: 53 · 2025: 95 · 2024: 93 · 2023: 67 · 2022: 46 · 2021: 18 · 2020: 1
- FWCI
- 3.16
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Journal of Advanced Research (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Natural Science Foundation of China (31172257, 31571600, 31271648)
- Science and Technology Innovation Talents in Universities of Henan Province (114100510008)
- Cotton Incorporated
- National Science Foundation (1658709)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Genetically Modified Organisms Research
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Keywords
- CRISPR/Cas9, genome editing, gene function, crop improvement, precision breeding, pathogen resistance, abiotic stress tolerance, plant development, secondary metabolism, fiber development, protospacer adjacent motif, off-target effects, Cas proteins, gene knock-in, gene knockout, epigenome editing
- MeSH terms
- Plant Breeding, Gene Editing, Agriculture, Humans, Phenotype, Stress, Physiological, Gene Targeting, Crops, Agricultural, Genome, Plant, Plants, Genetically Modified, Gene Knock-In Techniques, Agrobacterium, Plant Development, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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