Gene therapy development, re-engineering, and application to patients hold promise to revolutionize medicine, including therapies for disorders of the brain. Advances in delivery modalities, expression regulation, and improving safety profiles are of critical importance. Additionally, each inherited disorder has its own unique characteristics as to regions and cell types impacted and the temporal dynamics of that impact that are essential for the design of therapeutic design strategies. Here, we review the current state of the art in gene therapies for inherited brain disorders, summarizing key considerations for vector delivery, gene addition, gene silencing, gene editing, and epigenetic editing. We provide examples from animal models, human cell lines, and, where possible, clinical trials. This review also highlights the various tools available to researchers for basic research questions and discusses our views on the current limitations in the field.

The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.

As an emerging and innovative technology, gene-editing technology has been widely applied in crop breeding, human disease treatment, animal model research, drug and vaccine development, and microbial engineering. We mainly introduce the development of gene-editing technology, the application of clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) in Ganoderma lucidum breeding, the current challenges and optimization strategies in the use of gene-editing technology in Ganoderma breeding, as well as the current status of gene-editing technology in Ganoderma breeding. Finally, the future research directions and innovative strategies that gene editing may explore in Ganoderma breeding are prospects given the existing background, future research directions, and innovative strategies that gene editing may explore in Ganoderma breeding prospects.

Adenosine base editors (ABEs) facilitate A·T to G·C base pair conversion with significant therapeutic potential for correcting pathogenic point mutations in human genetic diseases, such as sickle cell anemia and β-thalassemia. Unlike CRISPR-Cas9 systems that induce double-strand breaks, ABEs operate through precise deamination, avoiding chromosomal instability. However, the off-target editing effects of ABEs remain inadequately characterized. In this study, we present a biochemical method Selict-seq, designed to evaluate genome-wide off-target editing by ABEs. Selict-seq specifically captures deoxyinosine-containing single-stranded DNA and precisely identifies deoxyadenosine-to-deoxyinosine (dA-to-dI) mutation sites, elucidating the off-target effects induced by ABEs. Through investigations involving three single-guide RNAs, we identified numerous unexpected off-target edits both within and outside the protospacer regions. Notably, ABE8e(V106W) exhibited distinct off-target characteristics, including high editing rates (>10%) at previously unreported sites (e.g. RNF2 and EMX1) and out-of-protospacer mutations. These findings significantly advance our understanding of the off-target landscape associated with ABEs. In summary, our approach enables an unbiased analysis of the ABE editome and provides a widely applicable tool for specificity evaluation of various emerging genome editing technologies that produce intermediate products as deoxyinosine.

With the increasing use of CRISPR-Cas9, detecting off-target events is essential for safety. Current methods primarily focus on guide RNA (gRNA) sequence mismatches, often overlooking the impact of DNA topology in regulating off-target activity. Here we present TOPO-seq, a high-throughput and sensitive method that identifies genome-wide off-target effects of Cas9 and base editors while accounting for DNA topology. TOPO-seq revealed that topology-induced off-target sites frequently harbor higher mismatches than the relaxed DNA sequence, with over 50% of off-target sites containing six mismatches, which are usually overlooked using previous methods. Applying TOPO-seq to three therapeutic gRNAs in hematopoietic stem cells identified 47 bona fide off-target loci, six of which are specifically induced by DNA topology. These findings highlight DNA topology as a regulator of off-target editing rates, establish TOPO-seq as a robust method for capturing DNA topology-induced off-target events and underscore its importance in off-target detection for developing safe genome-editing therapies.

Cas9 can cleave DNA in both blunt and staggered configurations, resulting in distinct editing outcomes, but what dictates the type of Cas9 incisions is largely unknown. In this study, we developed BreakTag, a versatile method for profiling Cas9-induced DNA double-strand breaks (DSBs) and identifying the determinants of Cas9 incisions. Overall, we assessed cleavage by SpCas9 at more than 150,000 endogenous on-target and off-target sites targeted by approximately 3,500 single guide RNAs. We found that approximately 35% of SpCas9 DSBs are staggered, and the type of incision is influenced by DNA:gRNA complementarity and the use of engineered Cas9 variants. A machine learning model shows that Cas9 incision is dependent on the protospacer sequence and that human genetic variation impacts the configuration of Cas9 cuts and the DSB repair outcome. Matched datasets of Cas9 and engineered variant incisions with repair outcomes show that Cas9-mediated staggered breaks are linked with precise, templated and predictable single-nucleotide insertions, demonstrating that a scission-based gRNA design can be used to correct clinically relevant pathogenic single-nucleotide deletions.

Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.1.

The CRISPR-Cas9 system has emerged as one of the most promising gene-editing technologies in biology. However, off-target effects remain a significant challenge. While recent advances in deep learning have led to the development of models for off-target prediction, these models often fail to fully leverage sequence pair information. Furthermore, as the models' parameter sizes increase, so do their complexities, limiting their practical applicability.

In this study, we introduce a novel multi-feature independent encoding method, which encodes the gRNA-DNA sequence pair into three distinct feature matrices to minimize information loss. Additionally, we propose a lightweight hybrid deep learning framework, CRISPR-MFH, that integrates multi-scale separable convolutions and hybrid attention mechanisms for efficient and accurate off-target prediction.

Extensive experiments across multiple benchmark datasets demonstrate that the proposed encoding method effectively captures critical features and that CRISPR-MFH outperforms or matches state-of-the-art models with significantly fewer parameters across multiple evaluation metrics.

This study offers a novel perspective for advancing deep learning technology in the realm of CRISPR-Cas9 off-target detection.

The gene editing field has advanced rapidly since the development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system because of its applicability in precisely modifying the genome. Among its multiple applications, the correction of genetic diseases has emerged as a potential curative treatment for many disorders that have eluded a cure to date. Despite its efficiency in achieving therapeutic levels of correction, the unexpected adverse effects of editing due to CRISPR/Cas9 nuclease activity are a major concern when translating these new strategies to the clinic. Multiple in silico tools and empirical methods have been developed to evaluate these off-target edits as well as other adverse alterations of the genome, including rearrangements, not only in ex vivo experiments but also in in vivo experiments. In this review, we summarize the available methods for the assessment of off-target effects of CRISPR/Cas9 systems, highlighting their advantages and limitations. Special attention will be paid to their application in pre-clinical studies and clinical trials, both in the manufacturing product and in the long-term follow-up of patients.

We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.

Genome editing enzymes can introduce targeted changes to the DNA in living cells 1-4 , transforming biological research and enabling the first approved gene editing therapy for sickle cell disease 5 . However, their genome-wide activity can be altered by genetic variation at on- or off-target sites 6-8 , potentially impacting both their precision and therapeutic safety. Due to a lack of scalable methods to measure genome-wide editing activity in cells from large populations and diverse target libraries, the frequency and extent of these variant effects on editing remains unknown. Here, we present the first population-scale study of how genetic variation affects the cellular genome-wide activity of CRISPR-Cas9, enabled by a novel, sensitive, and unbiased cellular assay, GUIDE-seq-2 with improved scalability and accuracy compared to the original broadly adopted method 9 . Analyzing Cas9 genome-wide activity at 1,115 on- and off-target sites across six guide RNAs in cells from 95 individuals spanning four genetically diverse populations, we found that variants frequently overlap off-target sites, with 13% significantly altering Cas9 editing activity by up to 33% indels. To understand common features of high-impact variants, we developed a new massively parallel biochemical assay, CHANGE-seq-R, to measure Cas9 activity across millions of mismatched target sites, and trained a deep neural network model, CHANGE-net, to accurately predict and interpret the effects of single-nucleotide variants on off-targets with up to six mismatches. Taken together, our findings illuminate a path to account for genetic variation when designing genome editing strategies for research and therapeutics.

The revolutionary CRISPR-Cas9 system leverages a programmable guide RNA (gRNA) and Cas9 proteins to precisely cleave problematic regions within DNA sequences. This groundbreaking technology holds immense potential for the development of targeted therapies for a wide range of diseases, including cancers, genetic disorders, and hereditary diseases. CRISPR-Cas9 based genome editing is a multi-step process such as designing a precise gRNA, selecting the appropriate Cas protein, and thoroughly evaluating both on-target and off-target activity of the Cas9-gRNA complex. To ensure the accuracy and effectiveness of CRISPR-Cas9 system, after the targeted DNA cleavage, the process requires careful analysis of the resultant outcomes such as indels and deletions. Following the success of artificial intelligence (AI) in various fields, researchers are now leveraging AI algorithms to catalyze and optimize the multi-step process of CRISPR-Cas9 system. To achieve this goal AI-driven applications are being integrated into each step, but existing AI predictors have limited performance and many steps still rely on expensive and time-consuming wet-lab experiments. The primary reason behind low performance of AI predictors is the gap between CRISPR and AI fields. Effective integration of AI into multi-step CRISPR-Cas9 system demands comprehensive knowledge of both domains. This paper bridges the knowledge gap between AI and CRISPR-Cas9 research. It offers a unique platform for AI researchers to grasp deep understanding of the biological foundations behind each step in the CRISPR-Cas9 multi-step process. Furthermore, it provides details of 80 available CRISPR-Cas9 system-related datasets that can be utilized to develop AI-driven applications. Within the landscape of AI predictors in CRISPR-Cas9 multi-step process, it provides insights of representation learning methods, machine and deep learning methods trends, and performance values of existing 50 predictive pipelines. In the context of representation learning methods and classifiers/regressors, a thorough analysis of existing predictive pipelines is utilized for recommendations to develop more robust and precise predictive pipelines.

Gene-edited animals are crucial for addressing fundamental questions in biology and medicine and hold promise for practical applications. In light of the rapid advancement of gene editing technologies over the past decade, a dramatically increased number of gene-edited animals have been generated. Genome editing at off-target sites can, however, introduce genomic variations, potentially leading to unintended functional consequences in these animals. So, there is an urgent need to systematically collect and collate these variations in gene-edited animals to aid data mining and integrative in-depth analyses. However, existing databases are currently insufficient to meet this need. Here, we present the Variation Database of Gene-Edited animals (VDGE, https://ngdc.cncb.ac.cn/vdge), the first open-access repository to present genomic variations and annotations in gene-edited animals, with a particular focus on larger animals such as monkeys. At present, VDGE houses 151 on-target mutations from 210 samples, and 115,710 variations identified from 107 gene-edited and wild-type animal trios through unified and standardized analysis and concurrently provides comprehensive annotation details for each variation, thus facilitating the assessment of their functional consequences and promoting mechanistic studies and practical applications for gene-edited animals.

DNA double-strand breaks (DSBs) are a major source of genomic instability. Physiological DSBs are naturally occurring breaks that happen during normal cellular processes. Unlike DNA breaks resulting from DNA damage due to external factors like radiation or chemicals, physiological DSBs play critical roles in various normal biological functions. Some key processes involving physiological DSBs include V(D)J recombination, transcription, and replication. These breaks are typically tightly controlled and are part of the cellular machinery designed to maintain and enhance genomic integrity and diversity. However, if these breaks are misrepaired or left unrepaired, they can contribute to genomic instability, potentially leading to senescence and diseases such as cancer. Here, we outline various methods commonly employed to detect physiological DSBs and introduce a detailed, step-by-step protocol for mapping these breaks using the in-suspension break labeling in situ and sequencing (sBLISS) technique. sBLISS offers single nucleotide resolution and is versatile enough to be applied to any cell type amenable to single-cell suspension. This comprehensive approach not only enhances our understanding of DSBs but also aids in the exploration of their roles in genomic instability.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a ground-breaking genome editing tool, which has revolutionized cell and gene therapies. One of the essential components involved in this system that ensures its success is the design of an optimal single-guide RNA (sgRNA) with high on-target cleavage efficiency and low off-target effects. This is challenging as many conditions need to be considered, and empirically testing every design is time-consuming and costly. In silico prediction using machine learning models provides high-performance alternatives.

We present CrisprBERT, a deep learning model incorporating a Bidirectional Encoder Representations from Transformers (BERT) architecture to provide a high-dimensional embedding for paired sgRNA and DNA sequences and Bidirectional Long Short-term Memory networks for learning, to predict the off-target effects of sgRNAs utilizing only the sgRNAs and their paired DNA sequences. We proposed doublet stack encoding to capture the local energy configuration of the Cas9 binding and applied the BERT model to learn the contextual embedding of the doublet pairs. Our results showed that the new model achieved better performance than state-of-the-art deep learning models regarding single split and leave-one-sgRNA-out cross-validations as well as independent testing.

The CrisprBERT is available at GitHub: https://github.com/OSsari/CrisprBERT.

Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology is a groundbreaking and dynamic molecular tool for DNA and RNA "surgery". CRISPR/Cas9 is the most widely applied system in oncology research. It is a major advancement in genome manipulation due to its precision, efficiency, scalability and versatility compared to previous gene editing methods. It has shown great potential not only in the targeting of oncogenes or genes coding for immune checkpoint molecules, and in engineering T cells, but also in targeting epigenomic disturbances, which contribute to cancer development and progression. It has proven useful for detecting genetic mutations, enabling the large-scale screening of genes involved in tumor onset, progression and drug resistance, and in speeding up the development of highly targeted therapies tailored to the genetic and immunological profiles of the patient's tumor. Furthermore, the recently discovered Cas12 and Cas13 systems have expanded Cas9-based editing applications, providing new opportunities in the diagnosis and treatment of cancer. In addition to traditional cis-cleavage, they exhibit trans-cleavage activity, which enables their use as sensitive and specific diagnostic tools. Diagnostic platforms like DETECTR, which employs the Cas12 enzyme, that cuts single-stranded DNA reporters, and SHERLOCK, which uses Cas12, or Cas13, that specifically target and cleave single-stranded RNA, can be exploited to speed up and advance oncological diagnostics. Overall, CRISPR platform has the great potential to improve molecular diagnostics and the functionality and safety of engineered cellular medicines. Here, we will emphasize the potentially transformative impact of CRISPR technology in the field of oncology compared to traditional treatments, diagnostic and prognostic approaches, and highlight the opportunities and challenges raised by using the newly introduced CRISPR-based systems for cancer diagnosis and therapy.

Comprehensive genome-wide studies are needed to assess the consequences of adeno-associated virus (AAV) vector-mediated gene editing. We evaluated CRISPR-Cas-mediated on-target and off-target effects and examined the integration of the AAV vectors employed to deliver the CRISPR-Cas components to neonatal mice livers. The guide RNA (gRNA) was specifically designed to target the factor IX gene (F9). On-target and off-target insertions/deletions were examined by whole-genome sequencing (WGS). Efficient F9-targeting (36.45% ± 18.29%) was apparent, whereas off-target events were rare or below the WGS detection limit since only one single putative insertion was detected out of 118 reads, based on >100 computationally predicted off-target sites. AAV integrations were identified by WGS and shearing extension primer tag selection ligation-mediated PCR (S-EPTS/LM-PCR) and occurred preferentially in CRISPR-Cas9-induced double-strand DNA breaks in the F9 locus. In contrast, AAV integrations outside F9 were not in proximity to any of ∼5,000 putative computationally predicted off-target sites (median distance of 70 kb). Moreover, without relying on such off-target prediction algorithms, analysis of DNA sequences close to AAV integrations outside the F9 locus revealed no homology to the F9-specific gRNA. This study supports the use of S-EPTS/LM-PCR for direct in vivo comprehensive, sensitive, and unbiased off-target analysis.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on-target (at the edited locus) and off-target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off-flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off-target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off-target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease-related variants by homology-directed repair. Off-flow identified a 3-base-pair-substitution and a mono-allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off-target missense variants and a mono-allelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On-target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off-flow. We used these results to filter out iPSC clones for subsequent functional experiments. Off-flow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off-flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.

Personalized medicine has transformed the treatment of cystic fibrosis (CF), providing customized therapeutic approaches based on individual genetic profiles. This review explores the genetic foundations of CF, focusing on mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and their implications for the development of the disease. The advent of genetic testing has enabled the association of specific mutations to disease severity, leading to the development of CFTR modulators like Ivacaftor, Lumacaftor, and Tezacaftor. Beyond CFTR mutations, genetic modifiers, including gene replacement therapy, genetic manipulation, lentivirus, and non-viral gene therapy formulations, along with environmental factors, play critical roles in influencing disease expression and outcomes. The identification of these modifiers is essential for optimizing therapeutic strategies. Emerging biomarkers, including inflammatory markers and pulmonary function indicators, aid in early disease detection and monitoring progression. Omics technologies are uncovering novel biomarkers, enabling more precise disease management. Pharmacogenomics has become integral to CF care, allowing for personalized approaches that consider genetic variations influencing drug metabolism, especially in antibiotics and anti-inflammatory therapies. The future of CF treatment lies in precision therapies, including CFTR modulators and cutting-edge techniques like gene therapy and CRISPR-Cas9 for mutation correction. As research evolves, these advances can improve patient outcomes while minimizing adverse effects. Ethical considerations and regulatory challenges remain critical as personalized medicine advances, ensuring equitable access and the long-term effectiveness of these innovative therapies.

CRISPR-based genome editing technologies have revolutionised the field of molecular biology, offering unprecedented opportunities for precise genetic manipulation. However, off-target effects remain a significant challenge, potentially leading to unintended consequences and limiting the applicability of CRISPR-based genome editing technologies in clinical settings. Current literature predominantly focuses on point predictions for off-target activity, which may not fully capture the range of possible outcomes and associated risks. Here, we present crispAI, a neural network architecture-based approach for predicting uncertainty estimates for off-target cleavage activity, providing a more comprehensive risk assessment and facilitating improved decision-making in single guide RNA (sgRNA) design. Our approach makes use of the count noise model Zero Inflated Negative Binomial (ZINB) to model the uncertainty in the off-target cleavage activity data. In addition, we present the first-of-its-kind genome-wide sgRNA efficiency score, crispAI-aggregate, enabling prioritization among sgRNAs with similar point aggregate predictions by providing richer information compared to existing aggregate scores. We show that uncertainty estimates of our approach are calibrated and its predictive performance is superior to the state-of-the-art in silico off-target cleavage activity prediction methods. The tool and the trained models are available at https://github.com/furkanozdenn/crispr-offtarget-uncertainty.

TCR-T cell therapy represents a promising advancement in adoptive immunotherapy for cancer treatment. Despite its potential, the development and preclinical testing of TCR-T cells face significant challenges. This review provides a structured overview of the key stages in preclinical testing, including in silico, in vitro, and in vivo methods, within the context of the sequential development of novel therapies. This review aimed to systematically outline the processes for evaluating TCR-T cells at each stage: from in silico approaches used to predict target antigens, assess cross-reactivity, and minimize off-target effects, to in vitro assays designed to measure cell functionality, cytotoxicity, and activation. Additionally, the review discusses the limitations of in vivo testing in animal models, particularly in accurately reflecting the human tumor microenvironment and immune responses. Performed analysis emphasizes the importance of these preclinical stages in the safe and effective development of TCR-T cell therapies. While current models provide valuable insights, we identify critical gaps, particularly in in vivo biodistribution and toxicity assessments, and propose the need for enhanced standardization and the development of more representative models. This structured approach aims to improve the predictability and safety of TCR-T cell therapy as it advances towards clinical application.

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology has revolutionized the field of genetic engineering, offering unprecedented potential for the targeted manipulation of DNA sequences. Advances in the mechanism of action of the CRISPR-Cas9 system allowed potential applicability for the treatment of genetic diseases. CRISPR-Cas9's mechanism of action involves the use of an RNA guide molecule to target-specific DNA sequences and the Cas9 enzyme to induce precise DNA cleavage. In the context of the CRISPR-Cas9 system, this review covers nonviral delivery methods for gene editing based on peptide internalization. Here, we describe critical areas of discussion such as immunogenicity, emphasizing the importance of safety, efficiency, and cost-effectiveness, particularly in the context of treating single-mutation genetic diseases using advanced editing techniques genetics as prime editor and base editor. The text discusses the versatility of cell-penetrating peptides (CPPs) in forming complexes for delivering biomolecules, particularly ribonucleoprotein for genome editing with CRISPR-Cas9 in human cells. In addition, it emphasizes the promise of combining CPPs with DNA base editing and prime editing systems. These systems, known for their simplicity and precision, hold great potential for correcting point mutations in human genetic diseases. In summary, the text provides a clear overview of the advantages of using CPPs for genome editing with CRISPR-Cas9, particularly in conjunction with advanced editing systems, highlighting their potential impact on clinical applications in the treatment of single-mutation genetic diseases. [Figure: see text].

Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present a protocol for establishing knockout cell clones by deletion of large gene fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays. This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines. For complete details on the use and execution of this protocol, please refer to Saito et al.,1 Saito and Endo et al.,2 and Higashi et al.3.

At the 8th International Workshop on Genotoxicity Testing meeting in Ottawa, in August 2022, a plenary session was dedicated to the genotoxicity risk evaluation of gene therapies, including insertional oncogenesis and off-target genome editing. This brief communication summarizes the topics of discussion and the main insights from the speakers. Common themes included recommendations to conduct tailored risk assessments based on a weight-of-evidence approach, to promote data sharing, transparency, and cooperation between stakeholders, and to develop state-of-the-art validated tests relevant to clinical scenarios.

Cells regularly repair numerous mutations. However, the effect of CRISPR/Cas9-induced dsDNA breaks on the repair processes of naturally occurring genome-wide mutations is unclear. In this study, we used TSCE5 cells with the heterozygous thymidine kinase genotype (TK+/-) to examine these effects. We strategically inserted the target sites for guide RNA (gRNA)/Cas9 and I-SceI into the functional allele and designed the experiment such that deletions of > 81 bp or base substitutions within exon five disrupted the TK gene, resulting in a TK-/- genotype. TSCE5 cells in the resting state exhibited 16 genome-wide mutations that affected cellular functions. After gRNA/Cas9 editing, these cells produced 859 mutations, including 67 high-impact variants that severely affected cellular functions under standard culture conditions. Mutation profile analysis indicated a significant accumulation of C to A substitutions, underscoring the widespread induction of characteristic mutations by gRNA/Cas9. In contrast, gRNA/Cas9-edited cells under conditions of S∼G2/M arrest and cyclin-dependent kinase 1 inhibition showed only five mutations. Transcriptomic analysis revealed the downregulation of DNA replication genes and upregulation of alternative DNA repair genes, such as zinc finger protein 384 (ZNF384) and dual specificity phosphatase, under S∼G2/M conditions. Additionally, activation of nucleotide and base excision repair gene, including O-6-methylguanine-DNA methyltransferase and xeroderma pigmentosum complementation group C, was observed. This study highlights the profound impact of CRISPR/Cas9 editing on genome-wide mutation processes and underscores the emergence of novel DNA repair pathways. Finally, our findings provide significant insights into the maintenance of genome integrity during genome editing.

Gene editing is a growing gene engineering technique that allows accurate editing of a broad spectrum of gene-regulated diseases to achieve curative treatment and also has the potential to be used as an adjunct to the conventional treatment of diseases. Gene editing technology, mainly based on clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein systems, which is capable of generating genetic modifications in somatic cells, provides a promising new strategy for gene therapy for a wide range of human diseases. Currently, gene editing technology shows great application prospects in a variety of human diseases, not only in therapeutic potential but also in the construction of animal models of human diseases. This paper describes the application of gene editing technology in hematological diseases, solid tumors, immune disorders, ophthalmological diseases, and metabolic diseases; focuses on the therapeutic strategies of gene editing technology in sickle cell disease; provides an overview of the role of gene editing technology in the construction of animal models of human diseases; and discusses the limitations of gene editing technology in the treatment of diseases, which is intended to provide an important reference for the applications of gene editing technology in the human disease.

The utilization of Streptomyces as a microbial chassis for developing innovative drugs and medicinal compounds showcases its capability to produce bioactive natural substances. Recent focus on the clustered regularly interspaced short palindromic repeat (CRISPR) technology highlights its potential in genome editing. However, applying CRISPR technology in certain microbial strains, particularly Streptomyces, encounters specific challenges. These challenges include achieving efficient gene expression and maintaining genetic stability, which are critical for successful genome editing. To overcome these obstacles, an innovative approach has been developed that combines several key elements: activation-induced cytidine deaminase (AID), nuclease-deficient cas9 variants (dCas9), and Petromyzon marinus cytidine deaminase 1 (PmCDA1). In this study, this novel strategy was employed to engineer a Streptomyces coelicolor strain. The target gene was actVA-ORF4 (SCO5079), which is involved in actinorhodin production. The engineering process involved introducing a specific construct [pGM1190-dcas9-pmCDA-UGI-AAV-actVA-ORF4 (SCO5079)] to create a CrA10 mutant strain. The resulting CrA10 mutant strain did not produce actinorhodin. This outcome highlights the potential of this combined approach in the genetic manipulation of Streptomyces. The failure of the CrA10 mutant to produce actinorhodin conclusively demonstrates the success of gene editing at the targeted site, affirming the effectiveness of this method for precise genetic modifications in Streptomyces.

Accurately identifying potential off-target sites in the CRISPR/Cas9 system is crucial for improving the efficiency and safety of editing. However, the imbalance of available off-target datasets has posed a major obstacle in enhancing prediction performance. Despite several prediction models have been developed to address this issue, there remains a lack of systematic research on handling data imbalance in off-target prediction. This article systematically investigates the data imbalance issue in off-target datasets and explores numerous methods to process data imbalance from a novel perspective. First, we highlight the impact of the imbalance problem on off-target prediction tasks by determining the imbalance ratios present in these datasets. Then, we provide a comprehensive review of various sampling techniques and cost-sensitive methods to mitigate class imbalance in off-target datasets. Finally, systematic experiments are conducted on several state-of-the-art prediction models to illustrate the impact of applying data imbalance solutions. The results show that class imbalance processing methods significantly improve the off-target prediction capabilities of the models across multiple testing datasets. The code and datasets used in this study are available at https://github.com/gzrgzx/CRISPR_Data_Imbalance.

Radiation cytogenetics has a rich history seldom appreciated by those outside the field. Early radiobiology was dominated by physics and biophysical concepts that borrowed heavily from the study of radiation-induced chromosome aberrations. From such studies, quantitative relationships between biological effect and changes in absorbed dose, dose rate and ionization density were codified into key concepts of radiobiological theory that have persisted for nearly a century. This review aims to provide a historical perspective of some of these concepts, including evidence supporting the contention that chromosome aberrations underlie development of many, if not most, of the biological effects of concern for humans exposed to ionizing radiations including cancer induction, on the one hand, and tumor eradication on the other. The significance of discoveries originating from these studies has widened and extended far beyond their original scope. Chromosome structural rearrangements viewed in mitotic cells were first attributed to the production of breaks by the radiations during interphase, followed by the rejoining or mis-rejoining among ends of other nearby breaks. These relatively modest beginnings eventually led to the discovery and characterization of DNA repair of double-strand breaks by non-homologous end joining, whose importance to various biological processes is now widely appreciated. Two examples, among many, are V(D)J recombination and speciation. Rapid technological advancements in cytogenetics, the burgeoning fields of molecular radiobiology and third-generation sequencing served as a point of confluence between the old and new. As a result, the emergent field of "cytogenomics" now becomes uniquely positioned for the purpose of more fully understanding mechanisms underlying the biological effects of ionizing radiation exposure.

Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.

OMEGA RNA (ωRNA)-guided endonuclease IscB, the evolutionary ancestor of Cas9, is an attractive system for in vivo genome editing because of its compact size and mechanistic resemblance to Cas9. However, wild-type IscB-ωRNA systems show limited activity in human cells. Here we report enhanced OgeuIscB, which, with eight amino acid substitutions, displayed a fourfold increase in in vitro DNA-binding affinity and a 30.4-fold improvement in insertion-deletion (indel) formation efficiency in human cells. Paired with structure-guided ωRNA engineering, the enhanced OgeuIscB-ωRNA systems efficiently edited the human genome across 26 target sites, attaining up to 87.3% indel and 62.2% base-editing frequencies. Both wild-type and engineered OgeuIscB-ωRNA showed moderate fidelity in editing the human genome, with off-target profiles revealing key determinants of target selection including an NARR target-adjacent motif (TAM) and the TAM-proximal 14 nucleotides in the R-loop. Collectively, our engineered OgeuIscB-ωRNA systems are programmable, potent and sufficiently specific for human genome editing.

The CRISPR/Cas9 system is a highly accurate gene-editing technique, but it can also lead to unintended off-target sites (OTS). Consequently, many high-throughput assays have been developed to measure OTS in a genome-wide manner, and their data was used to train machine-learning models to predict OTS. However, these models are inaccurate when considering OTS with bulges due to limited data compared to OTS without bulges. Recently, CHANGE-seq, a new in vitro technique to detect OTS, was used to produce a dataset of unprecedented scale and quality. In addition, the same study produced in cellula GUIDE-seq experiments, but none of these GUIDE-seq experiments included bulges. Here, we generated the most comprehensive GUIDE-seq dataset with bulges, and trained and evaluated state-of-the-art machine-learning models that consider OTS with bulges. We first reprocessed the publicly available experimental raw data of the CHANGE-seq study to generate 20 new GUIDE-seq experiments, and hundreds of OTS with bulges among the original and new GUIDE-seq experiments. We then trained multiple machine-learning models, and demonstrated their state-of-the-art performance both in vitro and in cellula over all OTS and when focusing on OTS with bulges. Last, we visualized the key features learned by our models on OTS with bulges in a unique representation.

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent β-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Pathogenic structure variations (SVs) are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements, focusing on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto-renal/branchio-oto syndrome. To address this, two CRISPR-based approaches were tested. First, we used Cas9 nuclease and paired gRNAs tailored to the patient's genome. The dual CRISPR-Cas9 system induced efficient correction of paracentric inversion in patient-derived fibroblast, and effectively restored the expression of EYA1 mRNA and protein, along with its transcriptional activity required to regulate the target gene expression. Additionally, we used CRISPR activation (CRISPRa), which leads to the upregulation of EYA1 mRNA expression in patient-derived fibroblasts. Moreover, CRISPRa significantly improved EYA1 protein expression and transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies could offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. Our findings demonstrate the potential of CRISPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.

The potential for off-target mutations is a critical concern for the therapeutic application of CRISPR-Cas9 gene editing. Current detection methodologies, such as GUIDE-seq, exhibit limitations in oligonucleotide integration efficiency and sensitivity, which could hinder their utility in clinical settings. To address these issues, we introduce OliTag-seq, an in-cellulo assay specifically engineered to enhance the detection of off-target events. OliTag-seq employs a stable oligonucleotide for precise break tagging and an innovative triple-priming amplification strategy, significantly improving the scope and accuracy of off-target site identification. This method surpasses traditional assays by providing comprehensive coverage across various sgRNAs and genomic targets. Our research particularly highlights the superior sensitivity of induced pluripotent stem cells (iPSCs) in detecting off-target mutations, advocating for using patient-derived iPSCs for refined off-target analysis in therapeutic gene editing. Furthermore, we provide evidence that prolonged Cas9 expression and transient HDAC inhibitor treatments enhance the assay's ability to uncover off-target events. OliTag-seq merges the high sensitivity typical of in vitro assays with the practical application of cellular contexts. This approach significantly improves the safety and efficacy profiles of CRISPR-Cas9 interventions in research and clinical environments, positioning it as an essential tool for the precise assessment and refinement of genome editing applications.

CRISPR-Cas technology has transformed our ability to introduce targeted modifications, allowing unconventional animal models such as pigs to model human diseases and improve its value for food production. The main concern with using the technology is the possibility of introducing unwanted modifications in the genome. In this study, we illustrate a pipeline to comprehensively identify off-targeting events on a global scale in the genome of three different gene-edited pig models. Whole genome sequencing paired with an off-targeting prediction software tool filtered off-targeting events amongst natural variations present in gene-edited pigs. This pipeline confirmed two known off-targeting events in IGH knockout pigs, AR and RBFOX1, and identified other presumably off-targeted loci. Independent validation of the off-targeting events using other gene-edited DNA confirmed two novel off-targeting events in RAG2/IL2RG knockout pig models. This unique strategy offers a novel tool to detect off-targeting events in genetically heterogeneous species after genome editing.

Genome editing involves precise modification of specific nucleotides in the genome using nucleases like CRISPR/Cas, ZFN, or TALEN, leading to increased efficiency of homologous recombination (HR) for gene editing, and it can result in gene disruption events via non-homologous end joining (NHEJ) or homology-driven repair (HDR). Genome editing, particularly CRISPR-Cas9, revolutionizes vaccine development by enabling precise modifications of pathogen genomes, leading to enhanced vaccine efficacy and safety. It allows for tailored antigen optimization, improved vector design, and deeper insights into host genes' impact on vaccine responses, ultimately enhancing vaccine development and manufacturing processes. This review highlights different types of genome editing methods, their associated risks, approaches to overcome the shortcomings, and the diverse roles of genome editing.

The CRISPR/Cas9 genome editing technology has transformed basic and translational research in biology and medicine. However, the advances are hindered by off-target effects and a paucity in the knowledge of the mechanism of the Cas9 protein. Machine learning models have been proposed for the prediction of Cas9 activity at unintended sites, yet feature engineering plays a major role in the outcome of the predictors. This study evaluates the improvement in the performance of similar predictors upon inclusion of epigenetic and DNA shape feature groups in the conventionally used sequence-based Cas9 target and off-target datasets. The approach involved the utilization of neural networks trained on a diverse range of parameters, allowing us to systematically assess the performance increase for the meticulously designed datasets- (i) sequence only, (ii) sequence and epigenetic features, and (iii) sequence, epigenetic and DNA shape feature datasets. The addition of DNA shape information significantly improved predictive performance, evaluated by Akaike and Bayesian information criteria. The evaluation of individual feature importance by permutation and LIME-based methods also indicates that not only sequence features like mismatches and nucleotide composition, but also base pairing parameters like opening and stretch, that are indicative of distortion in the DNA-RNA hybrid in the presence of mismatches, influence model outcomes.

Chimeric antigen receptor (CAR) T-cell therapy is a new and effective treatment for patients with hematologic malignancies. Clinical responses to CAR T cells in leukemia, lymphoma, and multiple myeloma have provided strong evidence of the antitumor activity of these cells. In patients with refractory or relapsed B-cell acute lymphoblastic leukemia (ALL), the infusion of autologous anti-CD19 CAR T cells is rapidly gaining standard-of-care status and might eventually be incorporated into frontline treatment. In T-ALL, however, leukemic cells generally lack surface molecules recognized by established CAR, such as CD19 and CD22. Such deficiency is particularly important, as outcome is dismal for patients with T-ALL that is refractory to standard chemotherapy and/or hematopoietic stem cell transplant. Recently, CAR T-cell technologies directed against T-cell malignancies have been developed and are beginning to be tested clinically. The main technical obstacles stem from the fact that malignant and normal T cells share most surface antigens. Therefore, CAR T cells directed against T-ALL targets might be susceptible to self-elimination during manufacturing and/or have suboptimal activity after infusion. Moreover, removing leukemic cells that might be present in the cell source used for CAR T-cell manufacturing might be problematic. Finally, reconstitution of T cells and natural killer cells after CAR T-cell infusion might be impaired. In this article, we discuss potential targets for CAR T-cell therapy of T-ALL with an emphasis on CD7, and review CAR configurations as well as early clinical results.

Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy.In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy.