Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.

Prediction of efficiencies for diverse prime editing systems in multiple cell types.

Applications of prime editing are often limited due to insufficient efficiencies, and it can require substantial time and resources to determine the most efficient pegRNAs and prime editors (PEs) to generate a desired edit under various experimental conditions. Here, we evaluated prime editing efficiencies for a total of 338,996 pairs of pegRNAs including 3,979 epegRNAs and target sequences in an error-free manner. These datasets enabled a systematic determination of factors affecting prime editing efficiencies. Then, we developed computational models, named DeepPrime and DeepPrime-FT, that can predict prime editing efficiencies for eight prime editing systems in seven cell types for all possible types of editing of up to 3 base pairs. We also extensively profiled the prime editing efficiencies at mismatched targets and developed a computational model predicting editing efficiencies at such targets. These computational models, together with our improved knowledge about prime editing efficiency determinants, will greatly facilitate prime editing applications.

Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

High-throughput PRIME-editing screens identify functional DNA variants in the human genome.

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and single-base resolution manner remains challenging. Here, we develop a pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp MYC enhancer via PRIME-mediated single-base resolution analysis. Next, we applied PRIME to functionally characterize 1,304 genome-wide association study (GWAS)-identified non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate that PRIME is capable of characterizing genetic variants at single-base resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.

Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need.

The c.1222C>T (p.Arg408Trp) variant in the phenylalanine hydroxylase gene (PAH) is the most frequent cause of phenylketonuria (PKU), the most common inborn error of metabolism. This autosomal-recessive disorder is characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Using real-world data, we observed that despite dietary and medical interventions, most PKU individuals harboring at least one c.1222C>T variant experience chronic, severe Phe elevations and do not comply with Phe monitoring guidelines. Motivated by these findings, we generated an edited c.1222C>T hepatocyte cell line and humanized c.1222C>T mouse models, with which we demonstrated efficient in vitro and in vivo correction of the variant with prime editing. Delivery via adeno-associated viral (AAV) vectors reproducibly achieved complete normalization of blood Phe levels in PKU mice, with up to 52% whole-liver corrective PAH editing. These studies validate a strategy involving prime editing as a potential treatment for a large proportion of individuals with PKU.

DNA base editing in nuclear and organellar genomes.

Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.

A Tale of Two Moieties: Rapidly Evolving CRISPR/Cas-Based Genome Editing.

Two major moieties in genome editing are required for precise genetic changes: the locator moiety for target binding and the effector moiety for genetic engineering. By taking advantage of CRISPR/Cas, which consists of different modules for independent target binding and cleavage, a spectrum of precise and versatile genome editing technologies have been developed for broad applications in biomedical research, biotechnology, and therapeutics. Here, we briefly summarize the progress of genome editing systems from a view of both locator and effector moieties and highlight the advance of newly reported CRISPR-conjugated base editing and prime editing systems. We also underscore distinct mechanisms of off-target effects in CRISPR-conjugated systems and further discuss possible strategies to reduce off-target mutations in the future.

CRISPR/Cas gene editing in the human germline.

The ease and efficacy of CRISPR/Cas9 germline gene editing in animal models paved the way to human germline gene editing (HGGE), by which permanent changes can be introduced into the embryo. Distinct genes can be knocked out to examine their function during embryonic development. Alternatively, specific sequences can be introduced which can be applied to correct disease-causing mutations. To date, it has been shown that the success of HGGE is dependent on various experimental parameters and that various hurdles (i.e. loss-of-heterozygosity and mosaicism) need to be overcome before clinical applications should be considered. Due to the shortage of human germline material and the ethical constraints concerning HGGE, alternative models such as stem cells have been evaluated as well, in terms of their predictive value on the genetic outcome for HGGE approaches. This review will give an overview of the state of the art of HGGE in oocytes and embryos, and its accompanying challenges.

PE-STOP: A versatile tool for installing nonsense substitutions amenable for precise reversion.

The rapid advances in genome editing technologies have revolutionized the study of gene functions in cell or animal models. The recent generation of double-stranded DNA cleavage-independent base editors has been suitably adapted for interrogation of protein-coding genes on the basis of introducing premature stop codons or disabling the start codons. However, such versions of stop/start codon-oriented genetic tools still present limitations on their versatility, base-level precision, and target specificity. Here, we exploit a newly developed prime editor (PE) that differs from base editors by its adoption of a reverse transcriptase activity, which enables incorporation of various types of precise edits templated by a specialized prime editing guide RNA. Based on such a versatile platform, we established a prime editing-empowered method (PE-STOP) for installation of nonsense substitutions, providing a complementary approach to the present gene-targeting tools. PE-STOP is bioinformatically predicted to feature substantially expanded coverage in the genome space. In practice, PE-STOP introduces stop codons with good efficiencies in human embryonic kidney 293T and N2a cells (with medians of 29% [ten sites] and 25% [four sites] editing efficiencies, respectively), while exhibiting minimal off-target effects and high on-target precision. Furthermore, given the fact that PE installs prime editing guide RNA-templated mutations, we introduce a unique strategy for precise genetic rescue of PE-STOP-dependent nonsense mutation via the same PE platform. Altogether, the present work demonstrates a versatile and specific tool for gene inactivation and for functional interrogation of nonsense mutations.

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

Editing VvDXS1 for the creation of muscat flavour in Vitis vinifera cv. Scarlet Royal.

Muscat flavour represents a group of unique aromatic attributes in some grape varieties. Biochemically, grape berries with muscat flavour produce high levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway, and VvDXS1 encodes the first enzyme in this plastidial pathway of terpene biosynthesis in grapevine. A single-point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has previously been identified as the major cause for producing muscat flavour in grapes. In this study, the same substitution in the VvDXS1 protein was successfully created through prime editing in the table grape Vitis vinifera cv. 'Scarlet Royal'. The targeted point mutation was detected in most of the transgenic vines, with varying editing efficiencies. No unintended mutations were detected in the edited alleles, either by PCR Sanger sequencing or by amplicon sequencing. More than a dozen edited vines were identified with an editing efficiency of more than 50%, indicating that these vines were likely derived from single cells in which one allele was edited. These vines had much higher levels of monoterpenes in their leaves than the control, similar to what was found in leaf samples between field-grown muscat and non-muscat grapes.

Prime editing: Mechanism insight and recent applications in plants.

Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.

Prime editing in plants: prospects and challenges.

Prime editors are reverse transcriptase (RT)-based genome-editing tools that utilize double-strand break (DSB)-free mechanisms to decrease off-target editing in genomes and enhance the efficiency of targeted insertions. The multiple prime editors that have been developed within a short span of time are a testament to the potential of this technique for targeted insertions. This is mainly because of the possibility of generation of all types of mutations including deletions, insertions, transitions, and transversions. Prime editing reverses several bottlenecks of gene editing technologies that limit the biotechnological applicability to produce designer crops. This review evaluates the status and evolution of the prime editing technique in terms of the types of editors available up to prime editor 5 and twin prime editors, and considers the developments in plants in a systematic manner. The various factors affecting prime editing efficiency in plants are discussed in detail, including the effects of temperature, the prime editing guide (peg)RNA, and RT template amongst others. We discuss the current obstructions, key challenges, and available resolutions associated with the technique, and consider future directions and further improvements that are feasible to elevate the efficiency in plants.

Targeted genomic translocations and inversions generated using a paired prime editing strategy.

A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.

Development of a highly efficient prime editor system in mice and rabbits.

The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.

Prime editing in plants and mammalian cells: Mechanism, achievements, limitations, and future prospects.

Clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system has revolutionized genetic research in the life sciences. Four classes of CRISPR/Cas-derived genome editing agents, such as nuclease, base editor, recombinase, and prime editor have been introduced for engineering the genomes of diverse organisms. The recently introduced prime editing system offers precise editing without many off-target effects than traditional CRISPR-based systems. Many researchers have successfully applied this gene-editing toolbox in diverse systems for various genome-editing applications. This review presents the mechanism of prime editing and summarizes the details of the prime editing system applied in plants and mammalian cells for precise genome editing. We also discuss the advantages, limitations, and potential future applications of prime editing in these systems. This review enables the researcher to gain knowledge on prime editing tools and their potential applications in plants and mammalian cells.

Precise genome engineering in Drosophila using prime editing.

Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.

Integrating Prime Editing and Cellular Reprogramming as Novel Strategies for Genetic Cardiac Disease Modeling and Treatment.

PURPOSE OF REVIEW: This review aims to evaluate the potential of CRISPR-based gene editing tools, particularly prime editors (PE), in treating genetic cardiac diseases. It seeks to answer how these tools can overcome current therapeutic limitations and explore the synergy between PE and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for personalized medicine. RECENT FINDINGS: Recent advancements in CRISPR technology, including CRISPR-Cas9, base editors, and PE, have demonstrated precise genome correction capabilities. Notably, PE has shown exceptional precision in correcting genetic mutations. Combining PE with iPSC-CMs has emerged as a robust platform for disease modeling and developing innovative treatments for genetic cardiac diseases. The review finds that PE, when combined with iPSC-CMs, holds significant promise for treating genetic cardiac diseases by addressing their root causes. This approach could revolutionize personalized medicine, offering more effective and precise treatments. Future research should focus on refining these technologies and their clinical applications.