The Next Stage of CRISPR: Programmable Insertion of Large DNA Fragments with QuadPE
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For decades, gene therapy faced a difficult engineering problem that needed to be solved: Precisely and efficiently inserting large DNA fragments — kilobases in size — at a desired genomic location without causing a double-strand break. Until now, all existing approaches either worked at low efficiency or were effective only in dividing cells. Naturepublished a groundbreaking study on 22 April 2026 in which Chinese scientists introduced a new method that overcomes this barrier: QuadPE (Quadruple pegRNA). Using four pegRNAs in coordination, this approach can insert DNA fragments ranging from 1.6 to 26 kilobases into the genome with up to 40% efficiency — even in nondividing cells. This advance has the potential to change the paradigm for treating monogenic diseases (single-gene disorders); it could have lasting implications across many fields, including oncology.
The Existing Problem: Why Is Inserting Large Gene Fragments Difficult?
Current genome-editing tools — CRISPR-Cas9, base editors, and prime editors (PE) — are powerful tools for correcting small mutations. However, each has its own limitations. In particular, inserting large DNA fragments (over 300 bases) remained an unsolved problem.
The fundamental problem with traditional approaches is that they rely on double-strand breaks (DSB — double-stranded break). These breaks are repaired through two pathways: NHEJ (non-homologous end joining) operates at approximately 1-5% efficiency and often produces uncontrolled insertions/deletions; HDR (homology-directed repair) is more precise but effective only in dividing cells. Many clinically important cell types — neurons, myocytes, hepatocytes — are nondividing or slowly dividing cells; HDR is of little use here.
KEY CONCEPTS — UNDERSTANDING QUADPE
CRISPR-Cas9: “Genetic scissors” that cut DNA by targeting specific genomic locations. Prime editing (PE): An advanced CRISPR derivative that can rewrite DNA without creating double-strand breaks; it makes a single-strand cut and encodes new sequences using reverse transcriptase. pegRNA: Prime editing guide RNA — a specialized RNA molecule that serves both as the guide identifying the target and as the template encoding the new sequence to be written in its place. epegRNA: An engineered, improved version of pegRNA. Integrase / Recombinase: Enzymes capable of integrating DNA into specific sites (e.g., used in PASTE and PASSIGE). Transposase (CAST): A DNA-integration system adapted from bacterial transposons.
Competitors in the Field: PASSIGE, PASTE, and CAST
In recent years, three main approaches were developed for inserting large DNA fragments into the genome, but each had serious limitations:
- Two-step process: first, a “landing pad” sequence is inserted
- Donor DNA is then integrated using BxbI integrase
- Efficiency <5% for a 9.5 kb insertion
- Uncertain in vivo safety profile
- Complex delivery (requires two proteins)
- An improved version of PASTE
- 3-26% efficiency for 1.6-3.5 kb insertions
- Efficiency falls to 1.5-6.6% at 9.5 kb
- Still requires a two-step process
- Integrase safety questions remain unresolved
- Bacterial Tn7-like transposase system
- Single step — an advantage
- Requires a large multisubunit protein complex
- Difficult delivery (large cargo)
- Efficiency at 9.5 kb is 1/12 of QuadPE's
QuadPE: The Elegant Choreography of Four pegRNAs
Developed by the team at Wuhan University, QuadPE overcomes the limitations of existing approaches through rational design. The central idea is simple but powerful: instead of a single pegRNA pair, four pegRNAs are used in coordination. Two of these pegRNAs mark the target genomic region, while the other two mark the ends of the donor DNA to be integrated. This creates complementary “3' flaps” (3'-ended tails) in both genomic and donor DNA. These complementary flaps pair with one another, enabling correct, directional, and precise integration.
QUADPE ARCHITECTURE — THE BASIC PRINCIPLE
The elegance of QuadPE lies in its lack of a need for double-strand breaks . Only single-strand cuts (nicks) are made. Two nicks on the genomic side and two on the donor side — four nicks in total. The complementary 3' flaps generated by these nicks interlock through sequence homology. DNA synthesis fills the gaps, and final repair steps complete the integration. The result: directional, precise, and controlled insertion of large gene fragments into the genome. No integrase, transposase, or dependence on the cell cycle is required.
The elegant choreography of complementary flaps: pairing of 3' flaps in genomic and donor DNA through sequence homology
Striking Results: Size Limits Are Being Overcome
The team's data are striking in many respects. QuadPE can successfully insert DNA fragments from 1.6 kb to 26 kb into HEK293T, K562, Huh-7, Jurkat, Hepa1-6, and mouse embryonic stem cells, as well as post-mitotic neurons and primary human T cells.
QuadPE's absolute efficiency advantage over PASTE, PASSIGE, and evoCAST
Which Settings Work Best? Optimization Findings
Detailed optimization studies underpin QuadPE's engineering success. The team's systematic screens revealed many critical parameters:
- gPAM-out is consistently superior — an average 1.7-fold higher efficiency
- Produces duplication between nick sites as an additional byproduct
- Mimics natural transposon-like target-site duplications (TSD)
- gPAM-in: deletion alongside insertion — suitable for genomic replacement
- gPAM-out is recommended for insertion alone
- 30 nucleotides is the optimal flap length
- 10 nt is too short — editing stops completely
- No additional benefit above 30 nt (plateau)
- 50% GC content provides the most stable performance
- 80% GC is variable depending on the locus
- Circular donor (cV6) > linear donor (LV4) for large fragments
The Most Important Advantage: Activity in Nondividing Cells
One of QuadPE's most revolutionary features is its independence from the cell cycle. The researchers demonstrated this in an elegant experiment: they arrested K562 cells in the G1 or G1/S phase using palbociclib (a CDK4/6 inhibitor — a drug also used in breast cancer) or thymidine. As expected, HDR-mediated insertion efficiency fell dramatically under these conditions — because HDR depends on the replication machinery of dividing cells. However, QuadPE largely maintained its efficiency.
More importantly, using cells isolated from mouse embryos at embryonic day 16-18.5, the team tested QuadPE in post-mitotic (no longer dividing) cortical neurons . Fragments of approximately 4.6 kb were shown to be inserted at multiple loci with up to 12.5% efficiency — a potential roadmap for gene therapy for neurological diseases. In addition, in primary human T cells, transfection with chemically modified epegRNAs and mRNA achieved, at the TRAC locus, 51% efficiency — offering great potential for CAR-T cell therapies.
Safety Profile: Minimal Off-Target Insertions
The most critical barrier to translating genome editing into clinical practice is off-target effects. The team assessed this issue carefully. By adapting the GUIDE-seq protocol, they performed an unbiased genome-wide analysis of off-target insertions. While on-target insertion was high at the AAVS1, TRAC, and VEGFA loci, off-target insertion events were negligible. Furthermore, at off-target loci predicted using CRISPOR, indel (insertion/deletion) rates remained below 1% .
An even more striking finding emerged from the team's Digenome-seq analysis: whereas standard Cas9 nuclease produced 29 off-target sites for TRAC gRNAs and 8 for VEGFA gRNAs, the Cas9 nickase (single-strand cutter) in QuadPE produced no detectable off-target breaks. This is an important safety advantage for QuadPE's clinical applicability.
What Does This Mean for Oncology and Medicine?
QuadPE has several important potential applications in cancer. First, CAR-T cell therapy: the study demonstrated DNA insertion at the TRAC locus in primary human T cells with 51% efficiency. This opens a route to producing more specific, safer, and potentially cheaper CAR-T products — because current CAR-T manufacturing relies on random integration and retroviral vectors, creating both safety and cost problems.
Second, hereditary cancer syndromes: for large deletions or point mutations in large genes such as BRCA1/2, TP53, APC, and Lynch syndrome genes, complete gene replacement using QuadPE is theoretically possible. Third, reinsertion of tumor suppressor genes: ex vivo reintroduction of large tumor suppressor genes lost in cancer cells could become a therapeutic avenue in the future. Fourth, hepatocyte-based gene therapies: the study demonstrated efficiency at the Rosa26 locus in Hepa1-6 mouse liver cells of over 55% — the liver is an important target organ for treating monogenic metabolic diseases.
The Road to Clinical Application: Still a Long Way to Go
A cautious assessment is essential, however. QuadPE has so far been tested only in cell culture and primary cells. Numerous barriers remain, including in vivo safety, long-term immunogenicity, delivery systems, and regulatory approvals. The following finding boxes summarize the stages of this journey:
🟢 ACHIEVEMENT: Cellular proof of concept
QuadPE has demonstrated high efficiency in 6 different cell lines and 2 primary cell types (mouse neurons + human T cells). This establishes the validity of the method's biological principle.
🟡 NEED 1: In Vivo Validation
Efficacy and safety must be tested in living organisms (mice, primates, and ultimately humans). Efficiency is expected to vary between organs (liver, muscle, neurons).
🟡 NEED 2: Delivery Systems
Effectively delivering four pegRNAs + PE protein + large donor DNA into living cells is a significant challenge. Various systems need to be developed, including AAV, lipid nanoparticles (LNP), and virus-like particles.
🔴 NEED 3: Long-Term Safety
An insertion event must not produce oncogenic or other pathological effects years later. Long-term monitoring of genomic stability and off-target effects is a prerequisite for clinical translation.
Implications for Turkish Practice and the Research Ecosystem
A technology such as QuadPE is strategically important to Türkiye's research and clinical ecosystem in several respects. First, research centers in Türkiye (the TÜBİTAK Genetic Engineering and Biotechnology Institute, Istanbul Technical University, Sabancı University, Koç University, METU, and the molecular biology laboratories of major medical centers) can rapidly adopt such emerging tools and integrate them into domestic research projects. The method published by the Wuhan University group in China has entered the literature under the principles of open science — opening the way for groups in Türkiye to implement it.
Second, specifically in cancer treatment, it represents an important opportunity for local CAR-T cell manufacturing programs . CAR-T cell therapies in Türkiye currently use imported products and are very expensive. Approaches similar to QuadPE could provide a foundation for local, safer CAR-T manufacturing in the future. Third, hereditary diseases prevalent in the Mediterranean region (thalassemia, sickle cell disease, SMA, familial hypercholesterolemia, BRCA1/2 founder mutations) could theoretically become candidates for large-fragment gene replacement. Finally, considering Türkiye's genetic disease profile , the relatively high prevalence of consanguineous marriage means that autosomal recessive single-gene disorders (monogenic diseases) are quite common, and these diseases fall precisely within the category targeted by QuadPE.
🎓 DROZDOGAN Academy — Commentary and Discussion
QUADPE · PRIME EDITING · LARGE GENE INSERTION · CAR-T · MONOGENIC DISEASES · ONCOLOGY CONNECTIONS
The Significance of This Advance
QuadPE offers an elegant solution to the question that followed the CRISPR revolution: “What can we do with large DNA?” Until now, gene therapy was mostly limited to “small changes” — correcting point mutations and adding/removing short sequences. Inserting large DNA fragments required either traditional recombinases (such as BxbI), transposons (Tn7-like), or complex two-step systems (PASTE/PASSIGE). QuadPE offers a single-step alternative that requires no double-strand breaks and works in nondividing cells. From an oncology perspective, this advance could open the door to applications such as safer and more precise CAR-T cell manufacturing, large gene replacement in hereditary cancer syndromes (BRCA, Lynch), and even ex vivo reinsertion of tumor suppressor genes. It is not yet clinical — but it is an important milestone at which the fundamental biological proof has been established.
Strengths
The study's most notable strength is its systematic, rigorous optimization approach. The team screened 24 different genome-donor combinations to identify the optimal design, then performed separate optimization studies for PE variants, flap length, GC content, and delivery formats. The results were confirmed in 6 different cell lines and at 14 different genomic loci — very strong evidence of generalizability. Validation in post-mitotic primary neurons and primary human T cells is also highly important for potential clinical application. Off-target analysis examined both GUIDE-seq-based insertion screening and Digenome-seq-based nickase activity — this two-pronged approach broadens the safety assessment. The use of long-read ONT and PacBio technologies provides molecular evidence that large fragments such as 9.5 kb inserts were correctly integrated. Direct comparisons with existing alternatives (PASTE, PASSIGE, evoCAST) demonstrate QuadPE's relative advantage in concrete numbers.
Limitations and Open Questions
Several critical points stand out in a critical assessment. First, all experiments were performed in cell culture or ex vivo primary cells — there are no in vivo data yet. Efficiency could fall dramatically in a living organism; organ-specific delivery challenges, in particular, may be major barriers. Second, a large cargo comprising four pegRNAs + PE protein + large donor DNA (1.6-26 kb) is incompatible with current delivery systems (AAV, LNP) — AAV capacity is limited to ~4.7 kb. This constitutes the largest practical barrier to the technology's clinical application. Third, the gPAM-out design produces 20-200 bp target-site duplications — this change in genomic structure could affect function in some genes (especially in regulatory regions); the authors describe it as resembling natural transposons, but its clinical effects are uncertain. Fourth, immunogenic responses were not evaluated — the development of neutralizing antibodies to the bacterial PE protein, especially with repeated administration, is a potential issue. Fifth, long-term genomic stability was not monitored; it is unknown whether changes occur at the insertion site weeks/months later. Sixth, the article's authors have filed a patent application for QuadPE (PCT/CN2025/143434), which increases the importance of independent validation studies. Finally, although Nature is prestigious, the study's publication date is very recent (22 April 2026); time is needed for independent groups to reproduce and validate it.
Implications for Clinical Practice
QuadPE is not a technology ready to enter clinical practice today — this is a critical point that must be emphasized. However, it carries several important messages for practitioners in oncology and clinical genetics. First, targeted insertion of large DNA fragments could become a clinical reality within the next 5-10 years; this will open new treatment avenues, especially in hereditary cancer syndromes (with ambitious ideas such as function-restoring repair in BRCA1/2-knockout tumors). Second, in CAR-T cell therapy, the standard may soon shift from current random integration to targeted integration — improving the safety and efficacy profile of CAR-T. Third, hematopoietic stem cell gene therapies (e.g., products such as CASGEVY for beta-thalassemia) are currently based on point-mutation correction; tools such as QuadPE could broaden this spectrum. Fourth, research centers in Türkiye (TÜBİTAK GMBE, major universities) may become interested in QuadPE-based preclinical studies — increasing domestic scientific capacity while laying the groundwork for future locally developed gene therapy products. Oncologists and clinical geneticists need to follow advances in this direction closely so that they can offer their patients appropriate treatment options in the future. The most important message is this: gene therapy is no longer a tool limited to “correcting point mutations”; it is increasingly becoming a sophisticated field of molecular engineering capable of large structural changes.
Questions for the Future
The critical questions for the coming period are: what will QuadPE's in vivo efficacy and safety look like in mouse models — might we see answers within the next 2-3 years? Can new delivery systems (modified AAV, LNP, virus-like particles) be developed for the cargo of four pegRNAs + large donor DNA? When integrated into existing CAR-T manufacturing protocols, will QuadPE measurably improve product quality and safety? Is in vivo germline gene replacement theoretically possible in hereditary cancer syndromes (BRCA1, Lynch, Li-Fraumeni) — and ethically acceptable? Can QuadPE enable “universal” gene replacement strategies covering multiple mutation types even within single-gene disorders? What will the profile look like when long-term (5+ years) follow-up data on off-target insertion and genomic stability become available? Specifically for Türkiye: could a locally developed QuadPE-based gene therapy product be created for conditions common in the Mediterranean region, such as thalassemia and sickle cell disease, and who should be responsible for the necessary infrastructure investments? The answers to these questions will shape the next decade of gene therapy and potentially the lives of millions of patients.
Scientific References
- Shi YJ, Ding ZY, Wu Y, He Z, Zhang YZ, Zhang YL, Zhang YM, Huang XR, Yin H, Zhang Y. Quadruple pegRNA enables programmable and efficient large genomic insertion. Nature. Published online: 22 April 2026. doi: 10.1038/s41586-026-10395-w
- Anzalone AV et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019;576:149–157. (Original prime editing article)
- Anzalone AV et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat Biotechnol 2022;40:731–740. (TwinPE)
- Wang J et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat Methods 2022;19:331–340.
- Yarnall MTN et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat Biotechnol 2023;41:500–512. (PASTE)
- Pandey S et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nat Biomed Eng 2025;9:22–39. (PASSIGE)
- Witte IP et al. Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase. Science 2025;388:eadt5199. (evoCAST)
- Zhang R et al. Amplification editing enables efficient and precise duplication of DNA from short sequence to megabase and chromosomal scale. Cell 2024;187:3936–3952.
- Doman JL et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 2023;186:3983–4002. (PE6 series)
- Gillmore JD et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N Engl J Med 2021;385:493–502. (Example of clinical CRISPR application)
This article was created using various editorial tools, including artificial intelligence, as part of the process. It was reviewed by human editors before publication.
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