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Revolutionizing Agriculture With CRISPR Technology: Applications, Challenges, and Future Perspectives.
CRISPR technologies are rapidly transforming agriculture by enabling precise and programmable modifications across a wide range of organisms. This review provides an overview of CRISPR applications in crops, livestock, aquaculture, and microbial systems, highlighting key advances in sustainable agriculture. In crops, CRISPR has accelerated the improvement of traits such as drought tolerance, nutrient efficiency, and pathogen resistance. In livestock and aquaculture, CRISPR has enabled disease-resistant pigs and poultry, hornless cattle, and fast-growing, stress-tolerant fish. Engineered microbes are also being leveraged to enhance nitrogen fixation and reduce input reliance. We examine the evolution of CRISPR tools, such as base and prime editing, multiplex editing, and epigenome modulation, that expand precision and control beyond traditional gene knockouts. These innovations offer significant advantages over conventional breeding, yet challenges remain, including off-target effects, delivery efficiency, and regulatory variability across countries. The review also explores emerging directions such as novel Cas variants and AI-integrated breeding platforms for high-throughput trait discovery. Together, these developments demonstrate the transformative potential of CRISPR technology to reshape agriculture, not only by enhancing productivity and resilience but also by reducing environmental impacts. With responsible implementation, CRISPR-enabled innovations are well-positioned to support global food security and sustainability targets by 2050.
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PAM-less plant genome editing using a CRISPR-SpRY toolbox.
The rapid development of the CRISPR-Cas9, -Cas12a and -Cas12b genome editing systems has greatly fuelled basic and translational plant research1-6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.
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Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor.
BACKROUND: CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system. METHODS: The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined. RESULTS: Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting. CONCLUSION: Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.
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Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.
Genome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly known as "scars." To address these issues, we developed a 2-stage, marker-assisted strategy to facilitate precise, "scarless" edits in Drosophila with a minimal requirement for molecular screening. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. We executed this 2-stage allele swap using a novel transformation marker that drives expression in the pupal wings, which can be screened for in the presence of common eye-expressing reporters. The tools we developed can be used to make a single change or a series of allelic substitutions in a region of interest in any D. melanogaster genetic background as well as in other Drosophila species.
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Prime Editing, CRISPR-Cas9, and NanoCas Genome Editing for Cancer Treatment.
Over the past two decades, genome editing has advanced dramatically from Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) to more refined systems such as CRISPR-Cas9, prime editing, and nanoCas technologies. These innovations have opened new frontiers in cancer treatment. This review aims to critically examine and compare recent advances in these genome editing platforms, with a focus on their molecular mechanisms, delivery challenges, oncological applications, and clinical prospects. We systematically explore how CRISPR-Cas9 enables gene knockouts, high-throughput functional genomic screens, and immune editing, while acknowledging its limitations due to off-target effects and genotoxicity. In contrast, base and prime editors offer precise, double-strand breaks (DSBs) free alternatives, suitable for correcting oncogenic mutations such as TP53, KRAS, and EGFR. Prime editing, although versatile, faces delivery and efficiency challenges. The emergence of nanoCas systems, derived from compact Cas orthologs, provides promising delivery advantages for in vivo applications. We also examine how tumor microenvironment, cell-type specificity, and immune barriers impact editing efficacy and safety. Strategies such as high-fidelity variants, optimized guide RNAs, and stimuli-responsive nanoparticles are discussed to enhance precision and minimize risk. Conclusively, integrating these genome editing tools into oncology requires addressing translational barriers while harnessing their precision and therapeutic potential for next-generation cancer treatments.
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Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System.
The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek's disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.
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Targeting a conserved functional motif in the PDS gene enables efficient CRISPR/Cas9 editing in banana.
Incomplete editing and chimeric phenotypes are major challenges in CRISPR/Cas9-mediated genome editing of polyploid crops. In this study, a single guide RNA (gRNA) was designed to target a conserved dinucleotide-binding motif within exon 3 of the phytoene desaturase (PDS) gene in 'Grand Naine' banana. The gRNA was carefully selected for GC content, guanine residues near the PAM, and predicted secondary structure to enhance Cas9 cleavage efficiency. Agrobacterium-mediated transformation of embryonic cell suspensions produced 102 putative transgenic plants, all exhibiting altered phenotypes, with 91% displaying albino and 9% pale green coloration, indicating efficient PDS gene knockout and absence of chimerism. Sequencing confirmed tri-allelic editing, with all edited plants consistently showing two identical and one distinct mutation. Notably, small in-frame deletions of two to six amino acids within the conserved motif were sufficient to abolish PDS function, confirming its critical role in carotenoid biosynthesis. This strategy is adaptable to clonally propagated polyploid crops, providing a practical framework for achieving high-efficiency, uniform genome edits and supporting the development of precise, non-chimeric CRISPR/Cas9 editing approaches.
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Base Editors-Mediated Gene Therapy in Hematopoietic Stem Cells for Hematologic Diseases.
Base editors, developed from the CRISPR/Cas system, consist of components such as deaminase and Cas variants. Since their emergence in 2016, the precision, efficiency, and safety of base editors have been gradually optimized. The feasibility of using base editors in gene therapy has been demonstrated in several disease models. Compared with the CRISPR/Cas system, base editors have shown great potential in hematopoietic stem cells (HSCs) and HSC-based gene therapy, because they do not generate double-stranded breaks (DSBs) while achieving the precise realization of single-base substitutions. This precise editing mechanism allows for the permanent correction of genetic defects directly at their source within HSCs, thus promising a lasting therapeutic effect. Recent advances in base editors are expected to significantly increase the number of clinical trials for HSC-based gene therapies. In this review, we summarize the development and recent progress of DNA base editors, discuss their applications in HSC gene therapy, and highlight the prospects and challenges of future clinical stem cell therapies.
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Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies.
Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of β-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.
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Genome editing in large animal models.
Although genome editing technologies have the potential to revolutionize the way we treat human diseases, barriers to successful clinical implementation remain. Increasingly, preclinical large animal models are being used to overcome these barriers. In particular, the immunogenicity and long-term safety of novel gene editing therapeutics must be evaluated rigorously. However, short-lived small animal models, such as mice and rats, cannot address secondary pathologies that may arise years after a gene editing treatment. Likewise, immunodeficient mouse models by definition lack the ability to quantify the host immune response to a novel transgene or gene-edited locus. Large animal models, including dogs, pigs, and non-human primates (NHPs), bear greater resemblance to human anatomy, immunology, and lifespan and can be studied over longer timescales with clinical dosing regimens that are more relevant to humans. These models allow for larger scale and repeated blood and tissue sampling, enabling greater depth of study and focus on rare cellular subsets. Here, we review current progress in the development and evaluation of novel genome editing therapies in large animal models, focusing on applications in human immunodeficiency virus 1 (HIV-1) infection, cancer, and genetic diseases including hemoglobinopathies, Duchenne muscular dystrophy (DMD), hypercholesterolemia, and inherited retinal diseases.
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