Efficient and precise genomic deletion in rice using enhanced prime editing.

Efficient and precise genomic deletion shows promise for investigating the function of proteins in plant research and enhancing agricultural traits. In this study, we tested the PRIME-Del (PDel) strategy using a pair of prime editing guide RNAs (pegRNAs) that targeted opposite DNA strands and achieved an average deletion efficiency of 55.8% for 60 bp fragment deletions at six endogenous targets. Moreover, as high as 84.2% precise deletion efficiency was obtained for a 2000 bp deletion at the OsGS1 site in transgenic rice plants. To add the bases that were unintentionally deleted between the two nicking sequences, we used the PDel/Syn strategy, which introduced multiple synonymous base mutations in the region that had to be patched in the RT template. The PDel/Syn strategy achieved an average of 58.1% deletion efficiency at six endogenous targets, which was higher than the PDel strategy. The strategies presented in this study contribute to achieving more accurate and flexible deletions in transgenic rice plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00153-9.

PE6c greatly enhances prime editing in transgenic rice plants.

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop breeding. Four new types of prime editors (PEs) named PE6a-d were recently generated using evolved and engineered reverse transcriptase (RT) variants from three different sources. In this study, we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice (Oryza sativa) plants. PE6c, with an evolved and engineered RT variant from the yeast Tf1 retrotransposon, yielded the highest prime-editing efficiency. The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes. We also demonstrated the feasibility of using two RT modules to improve prime-editing efficiency. Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants. In addition, our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

Assembly of Baculovirus Vectors for Multiplexed Prime Editing.

Efficient genome editing by using CRISPR technologies requires simultaneous and efficient delivery of multiple genetically encoded components to mammalian cells. Amongst all editing approaches, prime editing (PE) has the unique potential to perform seamless genome rewriting, in the absence of DNA double-strand breaks (DSBs). The cargo capacity required for efficient PE delivery to mammalian cells stands at odd with the limited packaging capacity of traditional viral delivery vectors. By contrast, baculovirus (BV) has a large synthetic DNA capacity and can efficiently transduce mammalian cells. Here we describe a protocol for the assembly of baculovirus vectors for multiplexed prime editing in mammalian cells.

Exploitation of enhanced prime editing for blocking aberrant angiogenesis.

INTRODUCTION: Aberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders. OBJECTIVES: The goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy. METHODS: An advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis. RESULTS: NILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology. CONCLUSION: DN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.

Developing a multi-modular assembled prime editing (mPE) system improved precise multi-base insertion efficiency in dicots.

INTRODUCTION: The Prime Editing (PE) system is a precise and versatile genome editing tool with great potential in plant breeding and plant synthetic biology. However, low PE efficiency severely restricts its application, especially in dicots. PE can introduce small tags to trace target protein or cis-element to regulate gene transcription which is an expertise superior to other gene editing tools. Owing to low efficiency, PE adaption in stably transformed Arabidopsis is lacking. OBJECTIVES: This study aimed to investigate the issue of low PE efficiency in dicots and develop systematic solutions to improve it. Currently, PE in dicots is undetectable and inconsistent, and this study seeks to address it. Split PE into several parts showed better performance in some target sites in mammal cells. We plan to discover the optimal split PE combination in dicot. METHODS: We conducted large-scale transformation experiments in dicot model plants Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by Agrobacterium-mediated transformation with deep amplicon sequencing (0.2-0.5 million clean total reads). RESULTS: The editing efficiency decreased upon using a fused reverse transcriptase (RT) or an extended pegRNA separately and further decreased dramatically when these were used together. With the help of the pol II strategy to express PE gRNA (pegRNA), we named the most effective split PE combination as a multi-modular assembled prime editing system (mPE). mPE exhibited improved precise editing efficiency on most gene sites with various editing types, ranging from 1.3-fold to 1288.5-fold and achieved PE on some sites that could not be edited by original PE2. Especially, mPE showed superiority for multi-base insertion with an average improvement of 197.9-fold. CONCLUSION: The original PE architecture strongly inhibited the cleavage activity of Cas9. Split PE improved PE efficiency extensively and was in favor of introducing small insertions in dicot plants, indicating that different PE variants might have their own expertise.

Emerging Perspectives on Prime Editor Delivery to the Brain.

Prime editing shows potential as a precision genome editing technology, as well as the potential to advance the development of next-generation nanomedicine for addressing neurological disorders. However, turning in prime editors (PEs), which are macromolecular complexes composed of CRISPR/Cas9 nickase fused with a reverse transcriptase and a prime editing guide RNA (pegRNA), to the brain remains a considerable challenge due to physiological obstacles, including the blood-brain barrier (BBB). This review article offers an up-to-date overview and perspective on the latest technologies and strategies for the precision delivery of PEs to the brain and passage through blood barriers. Furthermore, it delves into the scientific significance and possible therapeutic applications of prime editing in conditions related to neurological diseases. It is targeted at clinicians and clinical researchers working on advancing precision nanomedicine for neuropathologies.

Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges.

As the most versatile and precise gene editing technology, prime editing (PE) can establish a durable cure for most human genetic disorders. Several generations of PE have been developed based on an editor machine or prime editing guide RNA (pegRNA) to achieve any kind of genetic correction. However, due to the early stage of development, PE complex elements need to be optimized for more efficient editing. Smart optimization of editor proteins as well as pegRNA has been contemplated by many researchers, but the universal PE machine's current shortcomings remain to be solved. The modification of PE elements, fine-tuning of the host genes, manipulation of epigenetics, and blockage of immune responses could be used to reach more efficient PE. Moreover, the host factors involved in the PE process, such as repair and innate immune system genes, have not been determined, and PE cell context dependency is still poorly understood. Regarding the large size of the PE elements, delivery is a significant challenge and the development of a universal viral or nonviral platform is still far from complete. PE versions with shortened variants of reverse transcriptase are still too large to fit in common viral vectors. Overall, PE faces challenges in optimization for efficiency, high context dependency during the cell cycling, and delivery due to the large size of elements. In addition, immune responses, unpredictability of outcomes, and off-target effects further limit its application, making it essential to address these issues for broader use in nonpersonalized gene editing. Besides, due to the limited number of suitable animal models and computational modeling, the prediction of the PE process remains challenging. In this review, the fundamentals of PE, including generations, potential, optimization, delivery, in vivo barriers, and the future landscape of the technology are discussed.

[Prime-Editing Methods and pegRNA Design Programs].

It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A ↔ G and C ↔ T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.

Enrichment of prime-edited mammalian cells with surrogate PuroR reporters.

Prime editing is a programmable genetic method that can precisely generate any desired small-scale variations in cells without requiring double-strand breaks and DNA donors. However, higher editing efficiency is greatly desirable for wide practical applications. In this study, we developed a target-specific prime editing reporter (tsPER) and a universal prime editing reporter (UPER) to facilitate rapid selection of desired edited cells through puromycin screening. The modification efficiency of HEK3_i1CTT_d5G in HEK293T cells improved from 36.37 % to 64.84 % with the incorporation of tsPER. The target sequence of interested genes could be custom inserted into a selection cassette in tsPER to establish personalized reporters. The UPER demonstrated PE3 editing efficiency up to 74.49 % on HEK3_i1CTT_d5G and 73.52 % on HEK3_i1His6, achieved through co-selection with an additional pegRNA (puro) to repair the mutant PuroR cassette. Overall, tsPER and UPER robustly improved the efficiency of prime editing. Both of these approaches expand enrichment strategies for genomically modified cells and accelerate the generation of genetically modified models.

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.

Prime Editing and DNA Repair System: Balancing Efficiency with Safety.

Prime editing (PE), a recent progression in CRISPR-based technologies, holds promise for precise genome editing without the risks associated with double-strand breaks. It can introduce a wide range of changes, including single-nucleotide variants, insertions, and small deletions. Despite these advancements, there is a need for further optimization to overcome certain limitations to increase efficiency. One such approach to enhance PE efficiency involves the inhibition of the DNA mismatch repair (MMR) system, specifically MLH1. The rationale behind this approach lies in the MMR system's role in correcting mismatched nucleotides during DNA replication. Inhibiting this repair pathway creates a window of opportunity for the PE machinery to incorporate the desired edits before permanent DNA repair actions. However, as the MMR system plays a crucial role in various cellular processes, it is important to consider the potential risks associated with manipulating this system. The new versions of PE with enhanced efficiency while blocking MLH1 are called PE4 and PE5. Here, we explore the potential risks associated with manipulating the MMR system. We pay special attention to the possible implications for human health, particularly the development of cancer.

Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells.

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: "detection of targeted editing of CFTR in organoids").

Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice.

BACKGROUND: High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites. RESULTS: We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired. CONCLUSIONS: This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

Development and testing of a versatile genome editing application reporter (V-GEAR) system.

CRISPR-Cas9 and novel cas fusion proteins leveraging specific DNA targeting ability combined with deaminases or reverse transcriptases have revolutionized genome editing. However, their efficacy heavily relies upon protein variants, targeting single guide RNAs, and surrounding DNA sequence context within the targeted loci. This necessitates the need for efficient and rapid screening methods to evaluate these editing reagents and designs. Existing plasmid-based reporters lack flexibility, being fixed to specific DNA sequences, hindering direct comparisons between various editing approaches. To address this, we developed the versatile genome editing application reporter (V-GEAR) system. V-GEAR comprises genes detectable after desired editing via base editing, prime editing, or homology-directed repair within relevant genomic contexts. It employs a detectable synthetic cell surface protein (RQR8) followed by a customizable target sequence resembling genomic regions of interest. These genes allow for reliable identification of corrective editing and cell enrichment. We validated the V-GEAR system with base editors, prime editors, and Cas9-mediated homology-directed repair. Furthermore, the V-GEAR system offers versatility by allowing transient screening or stable integration at the AAVS1 safe harbor loci, rapidly achieved through immunomagnetic isolation. This innovative system enables direct comparisons among editing technologies, accelerating the development and testing of genome editing approaches.

Genome editing and kidney health.

Genome editing technologies, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas in particular, have revolutionized the field of genetic engineering, providing promising avenues for treating various genetic diseases. Chronic kidney disease (CKD), a significant health concern affecting millions of individuals worldwide, can arise from either monogenic or polygenic mutations. With recent advancements in genomic sequencing, valuable insights into disease-causing mutations can be obtained, allowing for the development of new treatments for these genetic disorders. CRISPR-based treatments have emerged as potential therapies, especially for monogenic diseases, offering the ability to correct mutations and eliminate disease phenotypes. Innovations in genome editing have led to enhanced efficiency, specificity and ease of use, surpassing earlier editing tools such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs). Two prominent advancements in CRISPR-based gene editing are prime editing and base editing. Prime editing allows precise and efficient genome modifications without inducing double-stranded DNA breaks (DSBs), while base editing enables targeted changes to individual nucleotides in both RNA and DNA, promising disease correction in the absence of DSBs. These technologies have the potential to treat genetic kidney diseases through specific correction of disease-causing mutations, such as somatic mutations in PKD1 and PKD2 for polycystic kidney disease; NPHS1, NPHS2 and TRPC6 for focal segmental glomerulosclerosis; COL4A3, COL4A4 and COL4A5 for Alport syndrome; SLC3A1 and SLC7A9 for cystinuria and even VHL for renal cell carcinoma. Apart from editing the DNA sequence, CRISPR-mediated epigenome editing offers a cost-effective method for targeted treatment providing new avenues for therapeutic development, given that epigenetic modifications are associated with the development of various kidney disorders. However, there are challenges to overcome, including developing efficient delivery methods, improving safety and reducing off-target effects. Efforts to improve CRISPR-Cas technologies involve optimizing delivery vectors, employing viral and non-viral approaches and minimizing immunogenicity. With research in animal models providing promising results in rescuing the expression of wild-type podocin in mouse models of nephrotic syndrome and successful clinical trials in the early stages of various disorders, including cancer immunotherapy, there is hope for successful translation of genome editing to kidney diseases.

Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.

Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.

Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa.

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases.

The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.

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.

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.