Emerging trends in prime editing for precision genome editing.

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.

From the Literature

Prime editing is a rapidly developing method of CRISPR/Cas-based genome editing. The increasing number of novel PE applications and improved versions demands constant analysis and evaluation. The present review covers the mechanism of prime editing, the optimization of the method and the possible next step in the evolution of CRISPR/Cas9-associated genome editing. The basic components of a prime editing system are a prime editor fusion protein, consisting of nickase and reverse transcriptase, and prime editing guide RNA, consisting of a protospacer, scaffold, primer binding site and reverse transcription template. Some prime editing systems include other parts, such as additional RNA molecules. All of these components were optimized to achieve better efficiency for different target organisms and/or compactization for viral delivery. Insights into prime editing mechanisms allowed us to increase the efficiency by recruiting mismatch repair inhibitors. However, the next step in prime editing evolution requires the incorporation of new mechanisms. Prime editors combined with integrases allow us to combine the precision of prime editing with the target insertion of large, several-kilobase-long DNA fragments.

Prime editing offers remarkable versatility in genome editing, but its efficiency remains a major bottleneck. While continuous optimization of the prime editing enzymes and guide RNAs (pegRNAs) has improved editing outcomes, the method of delivery also plays a crucial role in overall performance. To maximize prime editing efficiency, we implemented a series of systematic optimizations, achieving up to 80% editing efficiency across multiple loci and cell lines. Beyond integrating the latest advancements in prime editing, our approach combined stable genomic integration of prime editors via the piggyBac transposon system, selection of integrated single clones, the use of an enhanced promoter, and lentiviral delivery of pegRNAs, ensuring robust, ubiquitous, and sustained expression of both prime editors and pegRNAs. To further assess its efficacy in challenging cell types, we validated our optimized system in human pluripotent stem cells (hPSCs) in both primed and naïve states, achieving substantial editing efficiencies of up to 50%. Collectively, our optimized prime editing strategy provides a highly efficient and versatile framework for genome engineering in vitro, serving as a roadmap for refining prime editing technologies and expanding their applications in genetic research and therapeutic development.

Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here, we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.

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.

Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations, small insertions, and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA), which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs, especially when the genetic context precludes the screening of multiple pegRNAs, and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system, we generated other isogenic models of inherited retinal diseases (IRDs), including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure, such as plasmid concentrations, PE component ratios, and delivery method settings, showing that our improved workflow increased the hiPSC editing efficiency.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

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.

Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.

Prime editing of human hematopoietic stem cells has the potential to become a safe and efficient way of treating diseases of the blood directly in patients. By allowing site-targeted gene intervention without homology-directed repair donor templates and DNA double-stranded breaks, the invention of prime editing fuels the exploration of alternatives to conventional recombination-based ex vivo genome editing of hematopoietic stem cells. Prime editing is as close as we get today to a true genome editing drug that does not require a separate DNA donor. However, to adapt the technology to perform in vivo gene correction, key challenges remain to be solved, such as identifying effective prime editing guide RNAs for clinical targets as well as developing efficient vehicles to deliver prime editors to stem cells in vivo. In this review, we summarize the current progress in delivery of prime editors both in vitro and in vivo and discuss future challenges that need to be adressed to allow in vivo prime editing as a cure for blood disorders.

Prime editing enables precise genome modifications without DNA double-strand breaks, yet bacterial applications are limited by low efficiency and small edit sizes. Here, we develop PE-STAR, Prime Editing with SOS-Triggered and RecJ-Augmented Repair, to enhance prime editing in Escherichia coli. Removing three inhibitory 3'→5' exonucleases (SbcB, ExoX, and XseA) improved edited-strand retention, and extending post-transformation outgrowth increased editing efficiency. RecJ overexpression strengthened 5'-directed processing during flap resolution and gap expansion, biasing repair toward incorporation of the reverse-transcribed edited strand. To enrich edited cells, we integrated an SOS-responsive counter-selection circuit that links PE3-associated dual nicking to LexA-dependent gRNA expression targeting a plasmid encoding the toxin CcdB, thereby eliminating unedited cells. PE-STAR achieved up to 80%-90% editing efficiency for short-fragment modifications, representing up to 16-fold improvement across loci. The platform supported insertions, deletions, and replacements of up to 46 bp with high efficiency. Furthermore, installing an attB site by prime editing, followed by Bxb1 integrase recombination, enabled chromosomal integration of 3.2 and 8.0 kb cassettes with 100% recombination efficiency among screened colonies, including GFP reporter and riboflavin biosynthetic pathway. PE-STAR expands both the efficiency and functional scope of bacterial prime editing for programmable genome engineering.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing