Open research questions in CRISPR and Genetic Engineering

What the literature leaves open

• How cells choose among competing DNA double-strand break repair pathways, and whether the choice differs across the tree of life, remains poorly understood, limiting prediction of CRISPR editing outcomes, particularly in plants.

  5' Complementarity-Mediated End Joining (5'CMEJ) DNA repair · 2026 · DOI

• Future research in CRISPR–Cas9 plant biotechnology is expected to focus on expanding genome editing applications to orphan and underutilized crops, which are essential for regional food security but have historically received limited breeding investment (Figure 3). Advances in delivery technologies, particularly DNA-free and tissue culture-independent methods, are anticipated to broaden species applicability and improve regulatory acceptance. The integration of artificial intelligence and machine learning for sgRNA design, off-target prediction, and phenotype modeling will further enhance editing precision and efficiency. Additionally, emerging CRISPR variants and programmable epigenome editors offer new opportunities for reversible and environmentally responsive gene regulation. Collectively, these innovations are expected to strengthen the role of CRISPR–Cas9 in sustainable agriculture and accelerate the development of resilient, high-performing crop varieties (Somaly & Sokra, 2026; Somaly et al., 2026a; Somaly et al., 2026b). Figure 3. Application of CRISPR-Cas9 in plant science.

  CRISPR–Cas9 Applications in plant science: Advances, challenges, and future perspectives · 2026 · DOI

• In Sokra 1, 2 * Received: 10 December 2025/ Revised: 20 May 2026/ Accepted: 27 June 2026/ Published online: 25 July 2026 ©Journal of Agriculture and Technology 2026 1 Department of Soil and Crop Production, Faculty of Agronomy, University of Kratie, Cambodia 2 Department of Food Engineering, Faculty of Agro-Industry, University of Kratie, Cambodia *Corresponding author…

  CRISPR–Cas9 Applications in plant science: Advances, challenges, and future perspectives · 2026 · DOI

• However, a key limitation of dCAPS is that genomic target sites amenable to primer designs that both preserve PCR amplification and create recognition sites for inexpensive, high-performance restriction enzymes are scarce.

  OddCAPS: a simple, low-cost, universal technique for detecting single nucleotide variants · 2026 · DOI

• CRISPR-associated transposases (CAST) catalyze efficient RNA-guided DNA integration without double-strand breaks, yet their activity has not been established in plants.

  CRISPR-Associated Transposases Enable Programmable DNA Integration in Plants · 2026 · DOI

• While chromosomal injury has been considered the primary mechanism underlying pathogen killing by CRISPR-Cas antimicrobials, the synergistic role of gene disruption together with chromosomal injuries remains poorly understood.

  Synergistic CRISPR-Cas Antimicrobials through Essential and Defensive Gene Cotargeting in Staphylococcus aureus · 2026 · DOI

• 1. Lack of delivery system Animals can typically be systemically exposed to both modified and unmodified vectors. Exogenous CRISPR genes are protected from internal deterioration by modified vectors, but non-modified vectors may do so. The CRISPR/Cas system may alter normal tissues when it enters non-target cells, which could have unexpected effects. To lessen the entry of genes into unintended cells, a better delivery system, coated with biofilm or polypeptide to improve the onsite direct of foreign genes is required [51, 52, 53]. In response to alterations in the abrasive conditions in the target organ's microenvironment, gene delivery should be designed as environmentally friendly nanostructures. The nanostructures' core enters the cell through endocytosis after being first exposed by the breakdown of their outer shell in a particular microenvironment. An effective way to target infectious tissue on-site without affecting non-infectious tissues is needed that can be induced by various factors to release its genes [54]. 2. Off-Target Site Of action A specific area of the target site must be targeted by PAM sequences in order for many Cas proteins, including Cas9 and Cas12a. The PAM sequences are very specific to the CRISPR/Cas system type, and the use of this technology for therapeutic purposes is severely constrained in the absence of a specific PAM sequence close to the target region. These CRISPR tools need to be able to change to fit the targets they select in order to be more effective. Research has been done to create PAM variants of Cas9 and Cas12a that could recognize more than one PAM in order to accomplish this. Other essential Cas proteins, like Cas13 variants, also require designing to enhance their efficacy and selectivity [55, 56]. 3. RNA Destabilization and Mosaicism's Occurrence Due to RNase's widespread presence, RNA is prone to damage. It might significantly affect how well the CRISPR-based diagnostic system works. The occurrence of mosaicism, which occurs when transduced cells are used before the editing or cleavage of target nucleic acids, is another restriction on the use of the CRISPR system for therapeutic applications [60].

  A theragnostic approach to block the pathogenicity of coronavirus during crises of’ antimicrobial resistance · 2026 · DOI

• CRISPR/Cas9-induced DNA double-strand breaks (DSBs) are widely used for genome engineering, yet their capacity to provoke sister chromatid exchange (SCE) and associated genome instability remains incompletely understood, in part because exchanges between identical sister chromatids leave no sequence change and are invisible to conventional whole-genome sequencing.

  Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic targets · 2026 · DOI

• The CRISPR-Cas system, distinguished by its exceptional specificity and programmability, has rapidly evolved from a groundbreaking genome-editing technology into a powerful paradigm for molecular biosensing. Its applications have expanded beyond rapid pathogen detection to encompass the more Page 362 Tang et al. J Transl Genet Genom. 2026;10:343-68 challenging domain of early tumor screening. This evolution has been propelled in parallel by significant advances in device engineering, transitioning from conventional in-tube assays to integrated, closed microfluidic chips, and ultimately to portable platforms that combine precise temperature control, optical signal readout, and smartphone-based data processing. Collectively, these innovations have effectively miniaturized the “molecular laboratory” into a handheld format, greatly enhancing accessibility and usability[111-114]. Looking forward, the realization of fully automated and intelligent “sample-in, result-out” diagnostic systems remains a central objective. Achieving this goal will require the development of highly efficient sample preprocessing modules and multiplexed microfluidic architectures capable of sensitively detecting low-abundance targets and multidimensional biomarker signatures within complex biological matrices, as encountered in early-stage cancer screening[115-117]. In parallel, the integration of nanostructured materials with nucleic acid recognition elements offers promising solutions to long-standing challenges related to instrumentation cost, assay affordability, and signal stability[118-120]. Moreover, synergistic incorporation of artificial intelligence-both for optimizing sensing interfaces and for deciphering complex, high-dimensional signals-has the potential to substantially improve sensitivity, robustness, and multiplexing capacity. Despite these advances, several critical challenges must be addressed to enable widespread clinical implementation and point-of-care deployment. One of the foremost concerns is the risk of off-target activity, whereby CRISPR effectors may inadvertently recognize and cleave non-target sequences, leading to false-positive diagnostic results. Mitigation strategies include rigorous guide RNA design, engineering of high-fidelity Cas variants, and the application of computational tools for off-target prediction and optimization. In addition, integration barriers remain; although portability has improved, many current platforms still rely on specialized instrumentation or multi-step workflows, limiting their applicability in resource-constrained settings. Ongoing efforts to simplify operation through lyophilized reagents and user-friendly interfaces must also ensure reproducibility, long-term stability, and cost-effectiveness. Furthermore, comprehensive clinical validation and standardization are indispensable. While promising performance is frequently demonstrated under controlled laboratory conditions, robust evaluation using diverse, real-world clinical samples is essential to establish reliability and clinical relevance. Concurrently, evolving regulatory frameworks will necessitate stringent analytical and clinical validation to ensure safety and efficacy. Accordingly, future research should focus on several strategic directions. First, continuous optimization of Cas proteins and guide RNA architectures is essential to further enhance specificity and minimize off-target effects. Second, deeper integration of CRISPR-based assays with microfluidic technologies and consumer electronics is required to streamline workflows and enable truly decentralized diagnostics. Third, the establishment of standardized protocols, reference materials, and performance benchmarks will be crucial for accelerating regulatory approval and clinical translation. Finally, the exploration of novel Cas enzymes and advanced signal amplification strategies may broaden detection modalities and further improve sensitivity and multiplexing performance. In summary, CRISPR-based molecular biosensing stands at a pivotal crossroads, offering unprecedented opportunities to redefine disease diagnosis and health monitoring. By maintaining a balanced perspective that recognizes both its transformative potential and its technical and translational challenges, and by fostering sustained interdisciplinary collaboration among molecular biologists, engineers, clinicians, and regulatory scientists, this technology is well positioned to transition from bench to bedside, ushering in a new era of earlier, more accurate, and more accessible diagnostics. Tang et al. J Transl Genet Genom.

  Advances in CRISPR-based molecular biosensing: from pathogen detection to early tumor screening · 2026 · DOI

• Near-term progress in nanoparticle-enabled precision breeding is most likely to come from improved synthesis control, plantspecific targeting, better cargo stability, and reproducible RNAi formulations. Mid-term advances may include validated machinelearning models trained on plant-specific uptake and delivery datasets. Long-term possibilities such as autonomous AI-guided nanocarriers, DNA-origami logic systems, and organelle-specific genome editing should be presented as hypotheses rather than expected outcomes. Terms such as AGILE and NanoSafari should be defined in one sentence on first use and retained only if the underlying sources are primary and traceable. Future developments in nanocarrier enabled plant genome editing can be broadly divided into near term improvements and longer-term speculative directions. Near term progress is likely to focus on improving delivery efficiency, tissue specificity, and reproducibility. In contrast, concepts such as fully autonomous smart nanocarriers or integrated AI driven design platforms remain exploratory and will require substantial experimental validation. 8.1 Smart and AI-guided nanocarriers A major significant change in nanoparticle design is the shift from conducting experimental work through trial and error to using intelligent, data-driven approaches. Recently, Artificial Intelligence (AI) and Machine Learning (ML) have become very effective tools to rationalize nanocarrier formulation, predict biodistribution, and optimize design parameters (Eskandani, 2025). AI-powered platforms are being harnessed to screen through extensive nanomaterial libraries to derive structure-function relationships, thus reducing dependency on resource-intensive trial-and-error experiments (Eskandani, 2025). Structural data is also being analyzed using deep learning models to screen for high-affinity sequences (Dimple, 2025). An interesting development is the AI-Guided Ionizable Lipid Engineering (AGILE) platform which leverages Graph Neural Networks (GNNs) for the optimization of lipid nanoparticle formulations by varying features such as pKa and phospholipid chain length to enhance endosomal escape (Eskandani, 2025). Moreover, Large Language Model (LLM)-based platforms such as “NanoSafari” are leveraging the knowledge from more than 20,000 scientific publications to offer personalized suggestions for delivery systems (Eskandani, 2025). Besides, generative networks like Generative Adversarial Networks (GANs) are being considered for the creation of new nanocarrier designs with improved stability (Eskandani, 2025). On top of all that, the idea of “Digital Twins” in nanomedicine is becoming more popular. By integrating multi-omics data with cultivar-specific biomarkers, AI systems can customize delivery systems for each person (Eskandani, 2025).

  Programmable nanocarriers for precision plant engineering: converging nanotechnology, CRISPR, and next-generation breeding · 2026 · DOI

• CRISPR/Cas9 technology has significantly transformed the fields of genetics and genomics by providing a versatile and programmable platform for genome engineering. Its ability to introduce targeted genetic modifications has enabled the development of improved disease models, genetically modified organisms with desirable traits, and emerging therapeutic strategies for a variety of genetic disorders. Consequently, CRISPR-based genome editing has become an important tool in both fundamental biological research and translational biomedical applications. Despite these advances, several challenges remain that limit the widespread clinical implementation of CRISPR technologies. Ensuring the safety and precision of genome editing is essential, particularly in therapeutic contexts where unintended genomic alterations may have long-term biological consequences. Off-target mutations, insertion–deletion events (INDELs), and frameshift mutations continue to represent important technical challenges that require careful monitoring and methodological refinement. Recent developments in RNA-targeting CRISPR systems also highlight the expanding capabilities of genome engineering technologies. For example, programmable RNA acetylation systems based on CRISPR–Cas13, such as the engineered eNAT10– dCas13 fusion protein, have been developed to modulate RNA modifications, including N4-acetylcytidine (ac4C), 1 3Molecular Biology Reports (2026) 53:719 719 Page 14 of 20 s e c n e r e f e R ] 3 1 1, 1 1 1 [ d e t r o p e R s n o i t a t u m e v i t a n r e t l a () m e t s y s t e g r a t - ff O s n o i t a t u m e n e g r o, y c n e i c ffi e R D H, y c n e u q e r f l e d n i,. g. e ( s c i r t e m e m o c t u o, s e i t i l a d o m y r e v i l e d, s t e g r a t e n e g g n i d u l c n i, s n o i t a c i l p p a l a t n e m / i r e p x e 9 s a C R P S I R C d e t r o p e r f o y r a m m u s e v i s n e h e r p m o C 1 e l b a T s m e t s y s g n i s n o i t a t u m - t i d e e v i t a n r e t l A s n o i t a t i m i l l a t n e m i r e p x E d e v r e s b O d o h t e m y r e v i l e D t e g r a T s u c o l l e d o m e n e g t e g r a T l a t n e m i r e p x E e v i t c e j b O o N S.

  CRISPR/Cas9 in perspective: evaluating efficacy, delivery methods, and ethical challenges in genome editing · 2026 · DOI

• Advancements in genome editing, artificial intelligence, and nanotechnology are expected to further enhance gene therapy applications. The integration of these technologies may lead to more efficient, cost-effective, and widely accessible treatments in the future. 12. CONCLUSION Gene therapy represents a transformative approach in modern medicine by enabling targeted intervention at the genetic level. Although challenges such as safety, cost, and ethical considerations persist, continuous advancements in technology and regulatory frameworks are facilitating its clinical translation. With further research and innovation, gene therapy is expected to play a central role in the evolution of precision medicine and future healthcare systems. 13. REFERENCES 1. World Health Organization. Gene therapy overview. www.wjpr.net │ Vol 15, Issue 9, 2026. │ ISO 9001: 2015 Certified Journal │ 514 Taware. World Journal of Pharmaceutical Research 2. Doudna JA, Charpentier E. Genome editing with CRISPR-Cas9. Science., 2014. 3. Ginn SL, et al. Gene therapy clinical trials worldwide. J Gene Med., 2018. 4. Watson JD, Crick FH. Molecular structure of nucleic acids. Nature., 1953. 5. Anderson WF. Human gene therapy. Science, 1992. 6. Jinek M, et al. CRISPR-Cas9 system. Science, 2012. 7. Naldini L. Gene therapy returns to centre stage. Nature, 2015. 8. Wang D, et al. Viral vectors in gene therapy. Nat Rev Drug Discov., 2019. 9. Ramamoorth M, Narvekar A. Non-viral vectors. J Clin Diagn Res., 2015. 10. Dunbar CE, et al. Gene therapy applications. Science, 2018. 11. High KA, Roncarolo MG. Gene therapy benefits. N Engl J Med., 2019. 12. FDA. Gene therapy approvals report. 13. Ashley EA. Personalized medicine. Nat Rev Genet, 2016. 14. Hacein-Bey-Abina S, et al. Gene therapy risks. Science, 2003. 15. EMA/FDA guidelines on gene therapy. 16. National Institutes of Health. Future of gene therapy. www.wjpr.net │ Vol 15, Issue 9, 2026.

  GENE THERAPY: EMERGING TRENDS AND ITS TRANSFORMATIVE ROLE IN MODERN MEDICINE · 2026 · DOI

• appropriate governance this basis, we developed an overarching governance framework to coherently and comprehensively implement these recommendations and thereby mitigate biological threats, especially those involving SynBio-driven BW. that would enable improvements. On consensus-based According to our expert panel, the threat landscape of synthetic BW is increasingly shaped by the dual dynamics of “de-skilling” certain scientific tasks and their democratization, as has been previously discussed (Götting et al., 2025; National Academies of Sciences, Engineering, and Medicine, 2025; Peppin et al., 2025; Rose et al., 2024; Soice et al., 2023; Williams et al., 2025).

  Improving governance in the age of synthetic biology, artificial intelligence, and diverging threats · 2026 · DOI

• current NSABB empower diverse, the 2023). Furthermore, mobile applications for biosafety, biosecurity, and DU oversight may be helpful guides (IEGBBR International Experts Group of Biosafety and Biosecurity Regulators, 2024). Therefore, research institutions, funders, and other stakeholders should interdisciplinary, to review research institutional, and external committees proposals, evaluate potential harm, and determine suitable precautionary measures based on the progressing state of the art potentials and the advances in SynBio and other converging technologies. Research should be halted if its risks outweigh the benefits. Indeed, moratoria have been successfully installed in the past (Bonham, 2022), for instance, Asilomar (Berg et al., 1974), GOF research from 2014 to 2017 (White House Office of Science and Technology Policy, 2014; 2017), Chimera Research and human cloning (National Institutes of Health, 2015), as well as human genome editing (Lander et al., 2019). To complement these assessment mechanisms, we advocate for the enforcement of minimum cyberbiosecurity standards (Elgabry and Johnson, 2024; George, 2019; Greenbaum, 2023), including established frameworks, alongside mandatory digital signature requirements for gene library transactions to ensure supply chain integrity. In the context of SynBio and converging digital technologies, efforts to integrate cybersecurity principles into biosecurity are still in the early stages. Cyberbiosecurity practices are fragmented and only partially systematized in terms of communication, training, and policy development. It would be prudent to shift critical production capacities (such as vaccines) into distributed modular networks. This reduces single points of failure and increases response speed. Indeed, the WHO is establishing a network of distributed production capabilities with the mRNA Technology Transfer Hub in South Africa (WHO, 2025). At the same time, the WHO Pandemic Hub is building capacities for improved data and analytics flows, which are crucial for more resilient supply and production chains (WHO, 2024). Finally, we address the need for developing standardized interdisciplinary collaborative partnerships across cybersecurity and biosecurity domains to terminology fostering and facilitate more effective risk response strategies (Box 2). identification and coordinated 3.2.3 Adapt governance to keep pace with advances in SynBio through agile, adaptive and flexible governance measures Many governance approaches seem to remain trapped in the past. Contemporary governance continues to depend on antiquated regulations for governing a new emerging field of converging is technologies (Helbing and Ienca, 2024), which evidently inadequate to guarantee security in the upcoming decades. To ensure that regulations remain relevant, a proactive approach is necessary, one that is flexible enough to be quickly modified in response to scientific and technological advancements and emerging threats. It could involve the establishment of dedicated national task forces that monitor SynBio developments and recommend timely updates to biosecurity regulations, serving as a central point of coordination for all biosecurity stakeholders (Box 3). For example, the EU is currently exploring anticipatory governance models such as “regulatory sandboxes” and policy labs, already applied in AI policy, to faster adapt to innovation cycles (Ahern, 2025). The planned EU Biotech Act also proposes streamline and harmonize biotechnology regulation across the EU with integrated risk-based frameworks (EPRS, 2025). Policymakers should consider implementing a tiered regulatory approach, where different levels of oversight are applied based on the potential risk associated with specific SynBio applications. Engaging stakeholders from various sectors in the governance process can also enhance responsiveness to regulatory measures. life sciences. These should encompass Thorough biosecurity and biorisk management policies require the creation of tools and mechanisms to oversee fundamental and applied legislation, regulations, standards, guidelines, best practices, ethical codes, and research review procedures, as well as education and should be training. Governance complementary, mutually reinforcing, and encompass biosafety, biosecurity, and DU research.

  Improving governance in the age of synthetic biology, artificial intelligence, and diverging threats · 2026 · DOI

• The results presented should be considered in light of the limitations of this study. The Delphi method has been criticized for potential “homophily bias,” (Holeman et al., 2024) which we attempted to minimize by increasing participant diversity. For the group workshops, anonymity was waived for the benefit of the expert discussions as is common in Group Delphi processes (Niederberger and Renn, 2019). This may have caused various biases such as framing, anchoring, desirability bias, bandwagon effect, etc. (Winkler and Moser, 2016). We tried to reduce these biases utilizing multiple mixed breakout sessions during the workshops. However, the experts strongly voiced against it, preferring forum discussions. invite experts on Although we aimed to recruit participants with an active interest in this field, the study is limited by the willingness of those invited to participate. Since only biosecurity experts from German-speaking countries were included, perspectives of experts from other disciplines or regions were not included.

  Improving governance in the age of synthetic biology, artificial intelligence, and diverging threats · 2026 · DOI

• from non-governmental organizations, academic institutions, industry stakeholders, and citizen groups to inform policy development and strengthen democratic legitimacy in emerging technology governance. These advisory bodies would function as pivotal intermediaries between technical expertise (provided by scientists, risk assessors, and biosecurity specialists) and public interest considerations, including ethical, societal, and legal dimensions, thereby ensuring that governance decisions reflect broader societal values while maintaining scientific rigor. The European Biosecurity Regulators, which unite multiple European organizations to create joint guidance and training resources, may provide a working blueprint for multi-sector governance. In addition to this institutional approach, we advocate for the implementation of systematic transparency and open dialogue facilitate public engagement with complex initiatives technological developments, enabling discourse on the beneficial applications and potential misuse scenarios of SynBio, AI, and other converging technologies. that Such a multi-stakeholder advisory mechanism can significantly enhance public trust, improve policy effectiveness, and create more responsive governance systems capable of adapting to technological change (IAP, 2024; Moya et al., 2025). The integration of these approaches may offer a pathway toward more inclusive and legitimate governance frameworks that can better manage the DU nature of SynBio and emerging technologies while fostering innovation and protecting Public Health. These should be structured bottom-up on an institutional, regional, national and eventually international level (Box 1). 3.2.2 Improve biosecurity through robust training and monitoring systems The most pressing weaknesses in biosecurity concerning SynBio include training, poor monitoring of access authorizations, and a lack of risk awareness among personnel. To inadequate to individual, levels. Training mitigate these vulnerabilities, rigorous and conceptual biosecurity training programs and holistic monitoring systems must be established. This entails the implementation of exhaustive training programs, including interdisciplinary concepts, systems thinking, and collaborative methodologies, which must be regularly updated to reflect the latest advancements in SynBio and other converging technologies.

  Improving governance in the age of synthetic biology, artificial intelligence, and diverging threats · 2026 · DOI

• Scalability of the multiplexed epigenetic modulation approach to resource-limited settings in Africa and other endemic regions is mentioned but not addressed; the paper does not specify manufacturing protocols, cold-chain requirements, cost thresholds, or regulatory pathways needed to translate ligand-targeted nanoparticle and CAR-T delivery systems to low-income healthcare systems.

  Innovative CRISPR-Cas9 Strategies for Epigenetic Modulation of HIV-1 Latency: Targeting Tissue-Specific Reservoirs with Multiplexed Approach · 2026 · DOI

• The paper identifies ethical supervision for long-term impact monitoring and community engagement in gene-editing technologies as essential but does not specify which longitudinal biomarkers, follow-up intervals, or community consultation frameworks should be established to track unexpected off-target effects in HIV-positive populations receiving multiplexed CRISPR epigenetic therapies.

  Innovative CRISPR-Cas9 Strategies for Epigenetic Modulation of HIV-1 Latency: Targeting Tissue-Specific Reservoirs with Multiplexed Approach · 2026 · DOI

• The synergy between ligand-targeted nanoparticles and CAR-T cells for delivering multiplexed dCas9 effector proteins (KRAB, p300, LSD1, DNMT3A) to tissue-specific HIV-1 reservoirs has not been experimentally quantified; specific binding affinities, CAR-T activation thresholds, and in vivo biodistribution profiles for nanoparticle-CAR-T combinations require empirical characterization.

  Innovative CRISPR-Cas9 Strategies for Epigenetic Modulation of HIV-1 Latency: Targeting Tissue-Specific Reservoirs with Multiplexed Approach · 2026 · DOI

• The use of AI-optimized guide RNA libraries targeting heterogeneous viral reservoirs requires single-cell omics datasets stratified by tissue compartment, cell type, and latency state, but the paper does not identify which single-cell omics modalities (transcriptomics, chromatin accessibility, proteomics) are necessary or which publicly available HIV reservoir datasets contain adequate tissue representation for training multiplexed guide RNA algorithms.

  Innovative CRISPR-Cas9 Strategies for Epigenetic Modulation of HIV-1 Latency: Targeting Tissue-Specific Reservoirs with Multiplexed Approach · 2026 · DOI

• While base and prime editing techniques are noted as safer alternatives to double-strand break repair in CRISPR-mediated proviral excision, the paper does not specify which off-target genomic loci are most vulnerable to base/prime editing mutagenesis in HIV-infected cell populations or what validation threshold should confirm specificity before human application.

  Innovative CRISPR-Cas9 Strategies for Epigenetic Modulation of HIV-1 Latency: Targeting Tissue-Specific Reservoirs with Multiplexed Approach · 2026 · DOI

• The combination approach integrating dCas9 epigenetic editing with latency-reversing agents and broad-spectrum neutralizing antibodies lacks empirical validation of optimal dosing schedules, timing sequences, and synergistic thresholds needed to enhance immunosenescence while avoiding viral rebound across heterogeneous HIV-1 reservoirs.

  Innovative CRISPR-Cas9 Strategies for Epigenetic Modulation of HIV-1 Latency: Targeting Tissue-Specific Reservoirs with Multiplexed Approach · 2026 · DOI

• The paper identifies delivery efficiency and off-target effects as unresolved challenges for translating multiplexed CRISPR dCas9 epigenetic editing into clinical practice for HIV-1 latency reversal, but does not specify which tissue compartments (circulatory system, lymphoid organs, gastrointestinal tract, central nervous system) present the greatest delivery barriers or require distinct delivery optimization strategies.

  Innovative CRISPR-Cas9 Strategies for Epigenetic Modulation of HIV-1 Latency: Targeting Tissue-Specific Reservoirs with Multiplexed Approach · 2026 · DOI

• The EMSA binding kinetics (Kd determination) were measured only for PgiCas13b protein interacting with synthetic RNA substrates; systematic comparison of binding affinities and complex stability across different Cas13 orthologs (LbuCas13a, PsmCas13b, etc.) with their cognate crRNA sequences and variant leader-repeat hairpin designs is needed.

  A leader-repeat hairpin blocks extraneous CRISPR RNA production in diverse CRISPR-Cas13 systems · 2026 · DOI

• Northern blotting analysis was limited to detecting crRNA abundance in laboratory strains; direct quantification of hairpin-mediated suppression of off-target crRNA transcripts in natural CRISPR-Cas13 loci from diverse environmental isolates and evaluation of leader-repeat structural variations across different species has not been performed.

  A leader-repeat hairpin blocks extraneous CRISPR RNA production in diverse CRISPR-Cas13 systems · 2026 · DOI

Most-cited papers in CRISPR and Genetic Engineering

• Past, present, and future of CRISPR genome editing technologies · Cell · 2024 · 510 citations
• Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo · Nature Biotechnology · 2024 · 228 citations
• In vivo human T cell engineering with enveloped delivery vehicles · Nature Biotechnology · 2024 · 222 citations
• Improving prime editing with an endogenous small RNA-binding protein · Nature · 2024 · 220 citations
• Comprehensive review of CRISPR-based gene editing: mechanisms, challenges, and applications in cancer therapy · Molecular Cancer · 2024 · 210 citations
• Durable and efficient gene silencing in vivo by hit-and-run epigenome editing · Nature · 2024 · 173 citations
• Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing · Nature Biomedical Engineering · 2024 · 168 citations
• In vivo editing of lung stem cells for durable gene correction in mice · Science · 2024 · 167 citations
• Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA · Nature Communications · 2024 · 160 citations
• Rapid in silico directed evolution by a protein language model with EVOLVEpro · Science · 2024 · 157 citations

Most recent work

• Genomic in vitro transcription and Nanopore direct RNA sequencing of a human B-Lymphocyte cell line · bioRxiv · 2026
• Inhibition of TFIIH translocase reveals a novel lncRNA regulatory network at the BRCA1 locus · bioRxiv · 2026
• Highly effective gene inactivation in tetraploid Xenopus laevis with low-temperature-active engineered Cas12a · bioRxiv · 2026
• CRISPR/Cas12a: A Comprehensive Review from Structural Foundations to Applications in Nucleic Acid Precision Detection · Journal of Agricultural and Food Chemistry · 2026
• CRISPR-Cas12a biosensing technology advances and applications in precision diagnostics and cancer research · Talanta · 2026
• Target RNA-triggered CRISPR-Cas12a2 Preferentially Cleaves Collateral DNA over RNA · bioRxiv · 2026
• CRISPR/Cas12a-Based Electrogenerated Chemiluminescence Biosensing Platforms: Advancing Next-Generation Point-of-Care Testing · Analytical Chemistry · 2026
• Emerging synthetic biology-assisted technologies for overcoming antibiotic resistance: CRISPR-Cas, bacteriophage, microbiome, and metabolic engineering-based solutions · Journal of Microbiology · 2026
• More Options for Gene Editing in Hemoglobinopathies · New England Journal of Medicine · 2026
• A leader-repeat hairpin blocks extraneous CRISPR RNA production in diverse CRISPR-Cas13 systems · The EMBO Journal · 2026

Related topics in Biochemistry, Genetics and Molecular Biology