Evolution of Prime Editing: Enhancing Efficiency and Expanding Capacity

The potential to therapeutically alter the genome is one of the remarkable scientific developments in recent years. Genome editing technologies have provided an opportunity to precisely alter genomic sequence(s) in eukaryotic cells as a treatment option for various genetic disorders. These technologies allow the correction of harmful mutations in patients by precise nucleotide editing. Genome editing technologies such as CRISPR (clustered regularly interspaced short palindromic repeat) and base editors have greatly contributed to the practical applications of gene editing. However, these technologies have certain limitations, including imperfect editing, undesirable mutations, off-target effects, and lack of potential to simultaneously edit multiple loci. Recently, prime editing (PE) has emerged as a new gene editing technology with the potential to overcome the above-mentioned limitations. Interestingly, PE not only has higher specificity but also does not require double-strand breaks. In addition, a minimum possibility of potential off-target mutant sites makes PE a preferred choice for therapeutic gene editing. Furthermore, PE has the potential to introduce insertion and deletions of all 12 single-base mutations at target sequences. Considering its potential, PE has been applied as a treatment option for genetic diseases including hemoglobinopathies. β-Thalassemia, for example, one of the most significant blood disorders characterized by reduced levels of functional hemoglobin, could potentially be treated using PE. Therapeutic reactivation of the γ-globin gene in adult β-thalassemia patients through PE technology is considered a promising therapeutic strategy. The current review aims to briefly discuss the genome editing strategies and potential applications of PE for the treatment of β-thalassemia. In addition, the review will also focus on challenges associated with the use of PE.

CRISPR-Cas gene editing tools have revolutionized the field of biotechnology.Originally part of a bacterial immune system, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas) can cleave specific DNA sequences that match the RNA sequence coded into the Cas protein.CRISPR has great potential for curing genetic diseases and cancers.However, many challenges must be overcome, including the risk of off-target cuts and their resulting consequences, on-target effects, adverse immune system reactions, the need for a double-stranded DNA break (DSB), and the need for a donor template to carry out homology-directed repair.Alternatively, base editing and prime editing are technologies that can edit genes without a DSB and donor template.There is also a reduced risk of off-target editing, which mitigates a significant safety concern.A key application of CRISPR and other gene editing technologies is curing monogenic diseases, which arise from mutations in a single gene.This makes them simpler to treat than other types of genetic diseases.Here, we present the principles of CRISPR, base editing, and prime editing, and discuss how they can be applied in a clinical setting to treat or cure monogenic diseases.

Our review has described principles and functional importance of CRISPR-Cas9 with emphasis on the recent advancements, such as CRISPR-Cpf1, base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing and their potential applications in generating stress-tolerant plants. Improved agricultural practices and enhanced food crop production using innovative crop breeding technology is essential for increasing access to nutritious foods across the planet. The crop plants play a pivotal role in energy and nutrient supply to humans. The abiotic stress factors, such as drought, heat, and salinity cause a substantial yield loss in crop plants and threaten food security. The most sustainable and eco-friendly way to overcome these challenges are the breeding of crop cultivars with improved tolerance against abiotic stress factors. The conventional plant breeding methods have been highly successful in developing abiotic stress-tolerant crop varieties, but usually cumbersome and time-consuming. Alternatively, the CRISPR/Cas genome editing has emerged as a revolutionary tool for making efficient and precise genetic manipulations in plant genomes. Here, we provide a comprehensive review of the CRISPR/Cas genome editing (GE) technology with an emphasis on recent advances in the plant genome editing, including base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing (CRISPR-IGE), which can be used for obtaining cultivars with enhanced tolerance to various abiotic stress factors. We also describe tissue culture-free, DNA-free GE technology, and some of the CRISPR-based tools that can be modified for their use in crop plants.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR associated protein (CRISPR-Cas) has emerged and evolved as a revolutionary genome editing technology, transforming research across diverse biological disciplines. Over the past decade, this technology has unveiled numerous opportunities for precise genome manipulation. However, the processes of discovering Cas proteins, repurposing them as editing tools, selecting appropriate candidate tool from the CRISPR-toolbox, designing experiments, and analyzing data are often complex and require careful consideration. To support researchers at every stage of CRISPR experimentation, a wide array of web resources has been developed. In this article, we provide a comprehensive overview of standalone and web-based tools that assist in the identification of CRISPR-Cas systems and the design of guide RNAs (gRNAs). We also highlight tools for evaluating gRNA efficiency, predicting CRISPR-Cas9 mutation profiles, as well as tools for base editing and prime editing, and the analysis and visualization of experimental results. Additionally, we introduce CRISPR–Gateway for Accessing Tools and Resources (CRISPR-GATE), an all-inclusive web repository that consolidates publicly available tools for genome editing research. This repository offers a categorized and user-friendly interface, allowing researchers to quickly access relevant tools based on their specific needs. CRISPR-GATE aims to streamline the search for CRISPR resources, facilitating both education and accelerating innovation. The web repository can be accessed from https://crispr-gate.daasbioinfromaticsteam.in/.

The development of new adaptations of CRISPR-based genome editing platforms, such as base editing and prime editing, made it possible to broaden the scope and applications of genome editing in plants. First base editing and, more recently, prime editing evade the creation of double-stranded breaks in deoxyribonucleic acid (DNA) and the requirement of donor template of DNA for repair while enhancing editing efficiency and product purity over CRISPR/Cas9. As base-pair changes in genomic DNA determine many significant agronomic traits, crop varieties can be developed by precisely converting specific single bases in plant genomes. While base editing can introduce specific nucleotide changes, such as transition and transversion mutations in the targeted region, prime editing can create precise insertions, deletions, and all 12 types of point mutations using the “search-and-replace” method.This chapter provides the basic principles of base editing and prime editing technologies and their practical applications in plants. The chapter also summarizes the recent breakthroughs in applying base and prime editors in diverse plant species, including their use in improving disease resistance, herbicide resistance, nutritional quality, crop yield, and quality. Finally, this chapter aims to clearly understand base editing and prime editing in plants by outlining potential developments.

The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized the field of genetic engineering and molecular biology, offering unprecedented opportunities for precise genome editing. Initially discovered as a natural defense mechanism in bacteria, CRISPR-Cas9 technology enables targeted modifications of DNA, paving the way for significant advancements in gene therapy. This review article explores the multifaceted applications of CRISPR, focusing on its transformative potential in treating genetic diseases and various forms of cancer. We delve into the mechanisms by which CRISPR operates, emphasizing its dual-step process of targeting and cutting DNA, and highlighting its versatility in investigating gene functions and developing therapeutic strategies. Specific applications in genetic disorders, including sickle cell disease and cystic fibrosis, showcase the promising outcomes of ongoing pre-clinical and clinical trials. In the realm of oncology, CRISPR’s ability to edit oncogenes and enhance immune cell functions exemplifies its role in developing personalized cancer therapies. However, the rapid advancement of CRISPR technology also necessitates critical discussions around ethical considerations and regulatory challenges, particularly concerning off-target effects and the implications of germline editing. Looking ahead, innovations such as CRISPR 2.0, base editing, and prime editing are set to further expand the therapeutic landscape, enhancing the precision and safety of gene editing. This article underscores the importance of collaborative efforts across scientific, ethical, and regulatory domains to navigate the complexities of CRISPR technology while maximizing its benefits for patients and society.

The Aim of this article is to review CRISPR-based drug delivery systems and their therapeutic applications in gene therapy, focusing on advancements, challenges, and future prospects. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has emerged as a transformative tool in gene therapy, enabling precise and efficient genome editing with unprecedented ease compared to earlier platforms such as zinc finger nucleases and TALENs. By leveraging the Cas nuclease system, CRISPR enables targeted modification of disease-causing genes, offering promising therapeutic interventions for genetic disorders, cancers, and infectious diseases. However, the clinical translation of CRISPR-based therapeutics critically depends on the development of safe, efficient, and tissue-specific delivery systems. Current strategies encompass viral vectors (e.g., adeno-associated viruses, lentiviruses) and non-viral carriers (e.g., lipid nanoparticles, polymeric systems, cell-penetrating peptides), each with unique advantages and limitations in terms of delivery efficiency, cargo capacity, immunogenicity, and off-target effects. Advances in nanotechnology and biomaterials have further enabled targeted and stimuli-responsive delivery, enhancing therapeutic precision while minimizing systemic toxicity. Furthermore, CRISPR-based modalities such as base editing, prime editing, and CRISPR interference/activation expand the therapeutic landscape beyond simple gene knockout or repair. Despite remarkable progress, challenges remain in optimizing delivery to specific tissues, controlling off-target activity, and addressing ethical and regulatory concerns. This review provides a comprehensive overview of CRISPR-based drug delivery systems, recent preclinical and clinical advancements, and future perspectives in gene therapy, emphasizing innovations that bridge the gap between experimental success and clinical application.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base editing uses base editors to engineer precise changes for trait improvement. Newer technologies such as prime editing have now been developed as a “search-and-replace” tool to engineer all possible single-base changes. Owing to the availability of these, the field of genome editing has evolved rapidly to develop crop plants with improved traits. In this review, we present the evolution of the CRISPR/Cas system into new-age methods of genome engineering across various plant species and the impact they have had on tweaking plant genomes and associated outcomes on crop improvement initiatives.

β-Thalassemia is a prevalent inherited disorder of β-globin chains. The clustered regularly interspaced short palindromic repeats (CRISPR) genome editing system has emerged as a potential curative strategy. We conducted a bibliometric analysis to map global research trends in CRISPR-based thalassemia research. Original and review research articles were retrieved from the Scopus database using the search terms [TITLE-ABS-KEY (‘βeta thalassemia’ OR ‘β thalassemia’ OR thalassemia*) AND TITLE-ABS-KEY (‘gene edit*’ OR crispr* OR ‘clustered regularly interspaced short palindromic repeats’)] AND [LIMIT-TO (DOCTYPE, ‘re’) OR LIMIT-TO (DOCTYPE, ‘ar’)] for analysis. Bibliometric mapping and network visualization were performed using VOSviewer to analyze publication trends, authorship networks, international collaborations, keyword clusters, and citation metrics. Major CRISPR-based therapeutic strategies for thalassemia were reviewed to place experimental and clinical developments within a translational framework. The analysis demonstrates a clear transition from foundational genomic studies to translational applications, with leading contributions from the United States and China. Two dominant therapeutic strategies have emerged: direct correction of the HBB gene in hematopoietic stem cells and fetal hemoglobin reactivation via BCL11A repression. The latter strategy culminated in regulatory approval of exagamglogene autotemcel (Casgevy). Advances in base editing, prime editing, and strategies to improve engraftment are expected to enhance the precision and long-term efficacy of next-generation approaches. Clustered regularly interspaced short palindromic repeats-based research on thalassemia continues to expand, supported by extensive international collaboration and growing clinical translation. Future large-scale implementation will require advances in bioprocess engineering, cost reduction for ex vivo manufacturing, and adaptable treatment models for diverse healthcare systems.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Prime editing (PE), as a “search-and-replace” genome editing technology, has shown the attractive potential of versatile genome editing ability, which is, in principle, currently superior to other well-established genome-editing technologies in the all-in-one operation scope. However, essential technological solutions of PE technology, such as the improvement of genome editing efficiency, the inhibition of potential off-targets and intended edits accounting for unexpected side-effects, and the development of effective delivery systems, are necessary to broaden its application. Since the advent of PE, many optimizations have been performed on PE systems to improve their performance, resulting in bright prospects for application in many fields. This review briefly discusses the development of PE technology, including its functional principle, noteworthy barriers restraining its application, current efforts in technical optimization, and its application directions and potential risks. This review may provide a concise and informative insight into the burgeoning field of PE, highlight the exciting prospects for this powerful tool, and provide clues for questions that may propel the field forward.

Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.

Prime editing is an advanced genome editing technology that enables precise genetic modifications without inducing double-strand breaks or requiring donor DNA templates. Prime editing has rapidly become a versatile tool, supporting a wide range of genetic modifications, including point mutations, insertions and deletions. Here we examine the evolution of prime editing technologies, detailing advancements from the initial prime editing systems to recent innovations that enhance editing efficiency. Through structural modifications and improved delivery methods, prime editing has expanded its applicability across eukaryotic systems. By enabling access to previously challenging mutations, prime editing opens new avenues for therapeutic development and precision genetic research, with efficiency, specificity and accessibility expected to shape its future impact in genome engineering.

Precision medicine for sodium channelopathy-related autism and epilepsy.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is inarguably the most valuable gene editing tool ever discovered. Currently, three classes of CRISPR-based genome editing systems have been developed for gene editing, including CRISPR/CRISPR associate system (Cas) nucleases, base editors, and prime editors. Ever-evolving CRISPR technology plays an important role in medicine; however, the biggest obstacle to its use in clinical practice is the induction of off-target effects (OTEs) during targeted editing. Therefore, continuous improvement and optimization of the CRISPR system for reduction of OTEs is a major focus in the field of CRISPR research. This review aims to provide a comprehensive guide for optimization of the CRISPR-based genome editing system.