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Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator‐Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR‐associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost‐effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off‐target effects. Efforts are being made to reduce the off‐target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off‐target effects. Here, the current GE tools, the off‐target effects generated by GE technology, types of off‐target effects, mechanisms of off‐target effects, major concerns, and outcomes of off‐target effects in plants and animals are summarized. The methods to detect off‐target effects, tools for single‐guide RNA (sgRNA) design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized. Herein, the off‐target effects, types, mechanism, major concerns, and outcomes of off‐target effects in plants and animals are summarized. Moreover, methods to detect off‐target effects, tools for sgRNA design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized.

Summary Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C‐to‐T base editing in plants, we first compared seven cytidine deaminases in the BE3‐like configuration in rice. We found A3A/Y130F‐CBE_V01 resulted in the highest C‐to‐T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9‐mediated C‐to‐T base editing at A‐rich PAMs. To showcase its applications, we first applied A3A/Y130F‐CBE_V01 for multiplexed editing to generate microRNA‐resistant mRNA transcripts as well as pre‐mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F‐CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C‐to‐T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole‐genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome‐wide off‐target effects in rice. Different levels of cytidine deaminase‐dependent and sgRNA‐independent off‐target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome‐wide sgRNA‐dependent off‐target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1‐CBE_V04 and A3A/Y130F‐CBE_V04 as next‐generation plant CBEs with high editing efficiency, purity, and specificity.

Summary The CRISPR/Cas9 and Cas12a (Cpf1) tools have been used on a large scale for genome editing. A new effector with a single nuclease domain, a relatively small size, low‐frequency off‐target effects and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1). Cas12b has also shown temperature inducibility in mammalian systems. Therefore, this system is potentially valuable for editing the genomes of plant species, such as cotton, that are resistant to high temperatures. Using this new system, mutants of upland cotton were successfully generated following Agrobacterium‐mediated genetic transformation under a range of temperatures. Transformants (explants infected by Agrobacterium) exposed to 45 °C for 4 days showed the highest editing efficiency. No off‐target mutation was detected by whole‐genome sequencing. Genome edits by AacCas12b in T0 generation were faithfully passed to the T1 generation. Taken together, CRISPR/Cas12b is therefore an efficient and precise tool for genome editing in cotton plants.

Summary PAM‐relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome‐wide off‐target effects are largely unexplored. Here, we conduct whole‐genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9‐NGv1, Cas9‐NG, SpRY, nCas9‐NG‐PmCDA1, nSpRY‐PmCDA1 and nSpRY‐ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA‐dependent off‐target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off‐target mutations. Strikingly, ABE8e results in ~500 genome‐wide A‐to‐G off‐target mutations at TA motif sites per transgenic plant. ABE8e’s preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off‐target and background mutations occurring during PAM‐relaxed genome editing in plants.

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

Base editors, which enable targeted locus nucleotide conversion in genomic DNA without double‐stranded breaks, have been engineered as powerful tools for biotechnological and clinical applications. However, the application of base editors is limited by their off‐target effects. Continuously expressed deaminases used for gene editing may lead to unwanted base alterations at unpredictable genomic locations. In the present study, blue‐light‐activated base editors (BLBEs) are engineered based on the distinct photoswitches magnets that can switch from a monomer to dimerization state in response to blue light. By fusing the N‐ and C‐termini of split DNA deaminases with photoswitches Magnets, efficient A‐to‐G and C‐to‐T base editing is achieved in response to blue light in prokaryotic and eukaryotic cells. Furthermore, the results showed that BLBEs can realize precise blue light‐induced gene editing across broad genomic loci with low off‐target activity at the DNA‐ and RNA‐level. Collectively, these findings suggest that the optogenetic utilization of base editing and optical base editors may provide powerful tools to promote the development of optogenetic genome engineering. This study engineered the blue‐light‐activated base editors (BLBEs) by combining the photoswithes magnets with DNA deaminases. The BLBEs system showed a remarkable reduction in background editing and a significant enhancement of blue light‐dependent editing. Simultaneously, the potentially harmful off‐target effects on both the genomic and transcriptomic levels are greatly minimized.

ABE8e is a widely used adenine base editor that enables efficient A-to-G conversion in DNA across cellular and in vivo systems but is constrained by substantial off-target activity, which limits its therapeutic application.ABE8e induces widespread genome-wide off-target mutations, including events in oncogenes and tumor suppressor genes.Electrostatic remodeling yields a high-fidelity adenine base editor (erABE).erABE achieves near-background genome-wide off-target activity.A single dose of erABE achieves durable PCSK9 suppression and sustained low-density lipoprotein cholesterol reduction in mice.erABE increases low-density lipoprotein cholesterol uptake in human hepatic organoids via PCSK9 editing. Although gene editing offers durable therapeutic potential for genetic disorders such as hypercholesterolemia, widely used adenine base editors (ABEs) face translational obstacles due to potential off-target effects inherent in gene-editing systems. ABE8e has strong clinical potential owing to its high efficiency and rapid editing kinetics, but its genome-wide off-target effects remain poorly characterized. Here, we uncovered substantial genome-wide off-target edits of ABE8e using the GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) assay, including in oncogenes and tumor suppressors, highlighting significant risks for clinical translation. We therefore engineered a next-generation precision base editor, erABE, through electrostatic remodeling of the TadA8e deaminase. erABE reduces ABE8e’s genome-wide off-target activity by ~36.5-fold to background levels while retaining an efficiency indistinguishable from ABE8e. In mice, a single low-dose delivery of erABE packaged in lipid nanoparticles achieved robust Pcsk9 knockdown, resulting in a sustained, ~90% reduction in plasma PCSK9 protein and ~60% reduction in low-density lipoprotein cholesterol for at least 6 months. erABE also potently suppresses PCSK9 expression in human liver organoids. These results highlight the safety and durable therapeutic effects of erABE, supporting its translation as a clinical genome-editing platform. [Display omitted] Gene-editing technologies hold transformative promise for the treatment and prevention of human genetic diseases. Base editors, including cytosine base editors and adenine base editors, enable precise nucleotide conversion without generating double-strand breaks, thereby reducing genotoxic stress and expanding therapeutic applicability. However, permanent genomic modification necessitates exceptional fidelity, as even rare off-target events may have deleterious consequences, particularly if they arise in oncogenes or tumor suppressor genes. Accordingly, comprehensive assessment and rigorous optimization of editor safety are critical prerequisites for clinical translation. ABE8e is a widely adopted adenine base editor, valued for its robust activity across diverse cellular and in vivo contexts. Using the highly sensitive GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) platform revealed substantial genome-wide off-target mutagenesis. In this research, we implement an electrostatic remodeling strategy that reconfigures charge properties within noncatalytic regions of the ABE8e deaminase domain implicated in spontaneous DNA interactions. This approach yields a high-fidelity variant, erABE, which abolishes the genome-wide off-target effects while maintaining on-target efficiency comparable to the parental editor. These features position erABE as a safety-optimized candidate for clinical translation. Although currently engineered on an nCas9 (nickase Cas9) scaffold, the optimized TadA8e deaminase may be adaptable to alternative compact editor backbones, such as SakCas9 (SakKH) and IscB, thereby expanding its therapeutic versatility. While demonstrated here for low-density lipoprotein cholesterol reduction, this platform may also be extended to other monogenic disorders, including phenylketonuria and sickle cell disease. Electrostatic remodeling of Tad8e yields a high-precision adenine base editor (erABE) that eliminates genome-wide off-target effects while preserving editing efficiency and demonstrates therapeutic efficacy in mouse models and human liver organoids.

The failure of the cholesterol ester transfer protein (CETP) inhibitor, torcetrapib, has led to questions regarding whether the molecule itself or the mechanism of CETP inhibition was responsible for the adverse cardiovascular outcomes. Given the association with increases in blood pressure and plasma aldosterone levels, torcetrapib has been postulated to have adverse ‘off‐target’ effects. In this issue of British Journal of Pharmacology, Forrest and co‐workers have elegantly investigated these effects, demonstrating two salient points—(1) the pressor effect of torcetrapib is independent of CETP inhibition and (2) although associated with hyperaldosteronism, the pressor effect is likely not mediated by hyperaldosteronism. Anacetrapib, by contrast, did not demonstrate any pressor or off‐target effects. Despite these findings, it remains to be proven whether the adverse cardiovascular outcomes from torcetrapib were indeed related to the pressor effects and whether CETP inhibition by other agents will result in beneficial clinical outcomes. Yet, the studies of Forrest and co‐workers do bring us closer to unravelling the reasons behind the failure of torcetrapib. British Journal of Pharmacology (2008) 154, 1379–1381; doi:fn1; published online 9 June 2008

Gene editing stands for the methods to precisely make changes to a specific nucleic acid sequence. With the recent development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, gene editing has become efficient, convenient and programmable, leading to promising translational studies and clinical trials for both genetic and non-genetic diseases. A major concern in the applications of the CRISPR/Cas9 system is about its off-target effects, namely the deposition of unexpected, unwanted, or even adverse alterations to the genome. To date, many methods have been developed to nominate or detect the off-target sites of CRISPR/Cas9, which laid the basis for the successful upgrades of CRISPR/Cas9 derivatives with enhanced precision. In this review, we summarize these technological advancements and discuss about the current challenges in the management of off-target effects for future gene therapy.

The CRISPR system is a revolutionary genome editing tool that has the potential to revolutionize the field of cancer research and therapy. The ability to precisely target and edit specific genetic mutations that drive the growth and spread of tumors has opened up new possibilities for the development of more effective and personalized cancer treatments. In this review, we will discuss the different CRISPR-based strategies that have been proposed for cancer therapy, including inactivating genes that drive tumor growth, enhancing the immune response to cancer cells, repairing genetic mutations that cause cancer, and delivering cancer-killing molecules directly to tumor cells. We will also summarize the current state of preclinical studies and clinical trials of CRISPR-based cancer therapy, highlighting the most promising results and the challenges that still need to be overcome. Safety and delivery are also important challenges for CRISPR-based cancer therapy to become a viable clinical option. We will discuss the challenges and limitations that need to be overcome, such as off-target effects, safety, and delivery to the tumor site. Finally, we will provide an overview of the current challenges and opportunities in the field of CRISPR-based cancer therapy and discuss future directions for research and development. The CRISPR system has the potential to change the landscape of cancer research, and this review aims to provide an overview of the current state of the field and the challenges that need to be overcome to realize this potential.