Guest Column | August 10, 2026

The Second Holy Grail Of Gene Editing: Large Knock-Ins

By Steve Wolk, Ph.D., Founder & Chief Consultant, Sinawali Biotechnology Solutions

GettyImages-1316503044-dna-strand-double-helix-gene-editing

Gene editing has emerged as one of the most promising frontiers in modern medicine, but two major technical challenges continue to limit its full therapeutic potential. Solving either would dramatically expand the range of diseases that could be treated.

The first challenge is targeted delivery: reliably transporting gene-editing machinery to the specific organs and cell types where it is needed while minimizing off-target effects. As discussed in my previous Advancing RNA article, advances in delivery will determine how broadly gene-editing therapies can be applied across different tissues and diseases.

The second challenge is large knock-ins — the ability to precisely insert an entire gene or large exon into the genome. Achieving this capability would move gene editing beyond correcting small mutations and enable the replacement of large, disease-causing genetic sequences, opening new possibilities for treating a wide range of inherited disorders.

The Value Proposition

Genetic diseases originate from one or more mutations in the sequence that cause pathogenic changes to a protein. For example, diseases like cystic fibrosis or Duchene Muscular Dystrophy have hundreds or even thousands of pathogenic variants, so the feasibility of having an approved therapeutic for each variant of a disease quickly becomes untenable. The ability to replace an entire exon, or even an entire gene, to treat some or all of the variants with a single therapeutic, becomes a much more pragmatic solution. Current technologies to achieve such large knock-ins include viral approaches (gene therapy), prime editing variants, site-specific recombinases (SSRs), combining prime editing with integrases (PASTE and PASSIGE), transposons, and retrotransposons.

Despite the enormous potential upside of these approaches, a few possible complicating factors will need to be addressed once the technical hurdle has been addressed. For example, how straightforward is it to design the “perfect” wild type gene? Also, it is not well understood if there are secondary effects from the loss of certain mutations.

DNA Vs. RNA Approaches & Key Performance Parameters

Knocking in a large genetic payload requires two fundamental components – a molecular machine to do the cutting and pasting, and a template to provide the code be introduced. In principle, this template can be either DNA or RNA.

DNA Templates

The delivery of a double-stranded DNA (dsDNA) templates represents another unsolved technical problem, mainly due to nuclease degradation and the recognition of foreign DNA by immune system pathways such as cGAS-STING, ALRs, and TLR-9.

To date, the only technologies that have successfully evaded the immune response have been viral vectors, such as AAVs and lentiviruses. However, the viral approaches have their own inherent limitations, including liver toxicity, potential genotoxicity, template size limitations, and manufacturing challenges such as batch-to-batch reproducibility.

Solutions under development include closed end or end modified DNA (Syngoi, Generation Bio/XOMA Royalty, 4basebio) and circular ssDNA (cssDNA; e.g., Full Circle Therapeutics).29,43 Both approaches reduce immune reactions as well as susceptibility to exonuclease degradation.

RNA Templates

The continued complications with DNA delivery have driven the development of technologies that can utilize RNA templates such as retrotransposons and prime editing. Circularization of RNA is also being investigated (e.g., ORNA Tx/Lilly Therapeutics, Orbital TX/BMS), though additional development is needed for circularization efficiency, purification at large scale, IRES-based translation optimization.45

Performance

The performance of each technology for large knock-ins should be evaluated against efficiency parameters (size insertion range, insertion efficiency, % full length insertion, insertion to indel ratios (for CRISPR-based variants, performance across cell types), and safety toxicity parameters (off-target insertions, immune response/cytotoxicity).

Strategies For Large Knock-Ins

Prime Editing

Prime editing (PE) was first developed in David Lui’s lab.1 Prime editors contain a CRISPR complex fused to a reverse transcriptase, where the prime editing guide RNA (pegRNA) contains the new sequence to be inserted by the reverse transcriptase. Because the system, including the template sequence, is all RNA, problems associated with DNA templates are circumvented. Over the next few years, PE evolved through several generations (PE1 to PE7), which improved the insertion size to ~50 bps. Paired prime editing systems were then introduced such as twin-prime editing (TwinPE)2 which extended the size of the insert to ~400 base pairs with higher efficiencies.

Site Specific Recombinases (SSRs)

Background

There are a number of endogenous enzymes that rearrange DNA that can potentially be utilized/repurposed to specifically insert exons or entire genes of interest. These enzymes differ significantly in their mechanisms, specificity, and biological roles, and can be broadly classified into site-specific recombinases (SSRs) and transposases. SSRs include integrases, some recombinases, and resolvases. Traditional recombination-based approaches, such as the Cre-lox system, exhibit site specificity but lack programmability and guide RNA dependency.

Large Serine Recombinases (LSRs)

While capable of knocking in large payloads, success with known LSRs has been limited, with only ~5% efficiency and poor accuracy, often resulting in hundreds of off-target sites across the genome. Engineered LSRs represent a new way to precisely insert large DNA sequences into the genome that don’t require DNA cutting or rely on the cell’s repair machinery. Comprehensive engineering strategies that combine evolutionary screening (testing thousands of mutations to identify improvements), and machine-learning-guided optimization (including computational models to predict how combining mutations would affect performance, and programmable targeting) have generated promising new leads. The best variants achieved 97% specificity and efficiency up to 53%. The optimized enzymes also work across challenging cell types, achieving 33% efficiency in non-dividing cells, 24% in embryonic stem cells, and 17% in primary human T cells.14,30

Bridge Recombinases (bRNAs)

bRNAs, recently developed in Patrick Hsu’s lab, represent a new DNA editing technology to seamlessly write large changes into the right place in the human genome.10,20,36 The bRNAs have the potential to facilitate insertions, deletions, or inversions of up to one million base pairs in the genome. For example, the IS110 bridge recombination system contains two internal loops encoding nucleotide stretches that base-pair to the target DNA and the donor DNA, enabling insertion of DNA into genomic target sites, as well as programmable DNA excision and inversion.10

Early successes include correction of the disease-causing DNA repeats that cause Friedreich’s ataxia (which is a rare neurological disease), and the same approach could be applied to Huntington’s and other repeat expansion disorders.

Combinations Of PE And SSRs (PASTE & PASSIGE)

A hybrid approach for large knock-ins involves first using a PE variant to insert a recombination site into a genomic locus. Subsequently, a recombinase and a donor template are co-expressed to enable target integration. Two variants of this approach have been developed: PASTE (Programmable Addition via Site-specific Targeting Elements), developed in the Gootenberg/Abudayyeh lab, and PASSIGE (Prime-Assisted Site-Specific Integrase Gene Editing), developed in David Liu’s lab. These approaches have the potential to insert DNA fragments as large as 50,000 base pairs.22,31,34,37,48

Both PASTE and PASSIGE use a prime editor to insert a small landing site (46 base pairs) at a specific locus. A serine integrase, typically Bxb1, then inserts a large payload of DNA at this site. The lack of introduction of DSBs reduces indels and chromosomal rearrangements. The key difference is that PASTE utilizes a fused system (a CRISPR–Cas9 nickase fused to a reverse transcriptase (RT) and a serine integrase), while PASSIGE utilizes separate components (twin-prime editing to insert attP/B sites for Bxb1 binding, followed by DNA insertion using Bxb1 recombinase). Recent results suggest successful incorporation of payloads up to ~36 base pairs, with efficiencies ranging from 5% to 60% in human cells.

Transposons/Transposases

Background

Transposases are specialized enzymes that catalyze the excision, duplication, and reinsertion of DNA segments into new genomic locations. They are the driving force behind transposition (“jumping genes”) and therefore represent a major catalyst for genetic variability and genomic evolution. DNA Transposons operate via a cut-and-paste mechanism. The DNA element is excised from one site and inserted into another, typically not increasing in copy number. The enzymes, transposases, recognize terminal inverted repeats. Transposon-based technologies include piggyBac, Sleeping Beauty, and CRISPR-Transposases (CASTs).

The PiggyBac transposase recognizes transposon-specific inverted terminal repeats (ITRs) sequences located on both ends of the transposon vector and efficiently moves the contents from its original positions and integrates them into TTAA chromosomal sites.51 It has a very high efficiency, a high cargo capacity (>20 kb), and is seamless, meaning it leaves no footprint.51

The Sleeping Beauty transposase is a DNA transposon that typically favors TA dinucleotides, has a high efficiency but lower gene bias (e.g., more random), and leaves a 2-5 bp footprint.9

CASTs

CASTs are RNA-guided elements that integrate into DNA via a CRISPR mechanism, and are all evolutionarily derived from the Tn7 transposon. They achieve high specificity by working through a CRISPR mechanism, recognizing protospacer adjacent motifs (PAMs) and base-pairing to target protospacer sequences, but do not cause double stranded breaks.18,48 Metagenomi has developed a V-K CAST system that is expected to begin first in human trials soon.28 A variant dubbed evoCAST, developed via phage-assisted continuous evolution (PACE), has achieved up to 30% insertion in human cells.

Retrotransposons/Retrotransposases

Background

Retrotransposons, like retroviruses, move into genomes via RNA intermediates. Retrotransposons are highly abundant, existing in archaea, prokaryotes and eukaryotes. It has been estimated that ~42% of the human genome comes from transposable elements.4 Retrotransposons can exist as long tail repeat (LTR) versions, which are virus-like, and nonLTR versions (e.g., R2, LINEs/SINEs, often containing a poly-A tail). Non-LTR retrotransposons carry out insertion via target-primed reverse transcription (TPRT), which does not generate DSBs. TPRT has a copy-and-paste mechanism, and excels at multi-kb integrations into the highly conserved 28S rDNA loci. Therapeutic use of retrotransposons does not require a DNA template and therefore avoids the immune system response to DNA templates. These systems are also compatible with LNP delivery.

R2 Systems

The R2 element currently represents the dominant approach with biotechnology companies. They are non-LTR retrotransposons comprised of two essential components: the R2 protein, which contains DNA-binding motifs, domains for RNA-binding, RT, and endonuclease activity, and the R2 mRNA , which is flanked by synthetic 5’ and 3’ untranslated regions (UTRs) that are critical for the ordered cleavage process.5,48

Early work with R2 systems has yielded more than 10 kb knock-ins, with improving efficiencies, but the percent of full-length knock-ins was still low, and the specificity was variable. More recently, improvements have been made. For example, Typewriter Therapeutics has used retrotransposon technology for development of in vivo CAR-T therapies and liver-directed genetic medicines. Their construct is a two RNA system (an R2 mRNA and template RNA) that is engineered and optimized for transgene cassette insertion in the 28S ribosomal DNA via the TPRT mechanism. The resulting constructs showed strong expression in vitro in T cells and primary human hepatocytes and yielded 3-4% expression in vivo in rats when delivered in an LNP. Addition Therapeutics is working with a similar two RNA platform (dubbed PRINT, developed in Kathleen Collin’s lab at UC Berkeley), demonstrating delivery of GFP to rat, NHP, and human cells, with durable expression in vivo (rats and NHPs) for over one year with a single dose. Using this technology, Addition has generated preclinic data for obesity, Fabry disease, HIV protection, and wet AMD.

Summary: Current Leading Technologies

The promise of prime editing approaches continues to improve, and will likely gain traction for small insertions, up to a single exon (e.g., “classic” prime up to 50 bp, and dual systems like TwinPE for insertion up to up to 400 bp). For full gene knock-ins, progress for has been made for site-specific recombinases (LSRs, bridge recombinases), PE plus integrases/recombinases (PASTE & PASSIGE), transposons (CASTs, etc.), and retrotransposons. Retrotransposons maintain the appeal of an all-RNA system, avoiding the immune responses associated with DNA delivery.

These technologies are showing good potential, but complexity of the machinery creates challenges for manufacturing (consistency and cost) and delivery, as well as the development of better analytical methods. Viral/gene therapy has approved products, but the safety, manufacturing, and redosing concerns seem to be leading industry to look for alternative approaches.

In summary, there is no clear winner yet for large knock-ins. As is often the case, the final answer may be a spectrum of solutions, with a few of the technologies developing niches where they are favored. The final answer(s) will also depend on the new breakthroughs, which could significantly accelerate one or more of the technologies.

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Competing Interests:

The author has no financial interest in any of the companies discussed above.

About The Author:

Steve Wolk, Ph.D., is founder and chief consultant for Sinawali Biotechnology Solutions. He was formerly vice president of chemistry and Boulder site head for Editas Medicine, where he led the advanced technology, analytical sciences & structural biology, and process chemistry teams. In addition to an extensive history directing analytical groups for characterizing oligonucleotides, proteins, small molecules, and polymers, Wolk has led efforts to develop new technologies for delivery of CRISPR therapeutics and worked on an aptamer-based proteomic technology. He received his bachelor’s degree in chemistry from U.C. San Diego, where he received the Harold Urey Award. Wolk completed his Ph.D. work in biophysical chemistry at U.C. Berkeley under Ignacio Tinoco, Ph.D., where he used 2D-NMR and other spectroscopic techniques to characterize nonstandard DNA structures. Wolk also has broad experience in leadership, including management, developing future leaders, and creating/revising governance programs.