Transposase Gene Editing Shows High Efficiency in Biomanufacturing and Plant Breeding

Transposase-based gene editing systems are demonstrating significant advantages over CRISPR-Cas9 in both biopharmaceutical manufacturing and plant breeding applications, according to recent research and industry developments. The technology, based on "jumping genes" discovered by Nobel laureate Barbara McClintock, is addressing key limitations of traditional gene editing approaches.

In biopharmaceutical manufacturing, transposon systems such as Leap-In Transposase and piggyBac transposase have been adopted for engineering Chinese Hamster Ovary (CHO) cells, which have served as the primary mammalian host for biopharmaceutical production since the 1980s. The first CHO-derived product was approved by the FDA in 1987. Transposases offer two critical advantages over targeted nucleases: multi-copy integrations across the CHO genome and high transposition efficiency, resulting in highly productive homogeneous cell populations.

The transposase enzyme typically requires only a short target sequence (e.g., TTAA) and an open chromatin region, resulting in anywhere between 2 and 50 integration copies per CHO genome. The integrity of the transposed DNA is stably maintained at every integration site. This contrasts sharply with CRISPR-Cas9 approaches, which face low insertion efficiencies in industrially relevant CHO cells, estimated to be around 1 in 100,000.

Traditional gene editing approaches have faced significant limitations. Homologous recombination is a rare event in mammalian cells, occurring with a frequency of approximately 1 in 106-107 cells per generation. CHO cells are particularly recalcitrant to homology dependent repair (HDR), making this approach unviable for CHO engineering. While targeted nucleases like CRISPR-Cas9, TALENs, and ZFNs sought to address these limitations by inducing site-specific double-strand breaks at defined genomic loci, non-homologous end joining (NHEJ), the dominant repair pathway in mammalian cells, quickly ligates broken DNA ends, resulting in insertions or deletions.

A peer-reviewed study in Molecular Therapy: Methods & Clinical Development used optical genome mapping (OGM) to assess genomic alterations from multiple gene editing approaches, including transposons, lentiviral transduction, and CRISPR-Cas9 locus insertion. The study reported that OGM was used as a genome-wide, unbiased method to detect large genomic rearrangements and structural variants with sensitivity to variant allele fractions as low as 5% in human induced pluripotent stem cell lines before and after gene editing.

Researchers found that transposons and lentiviral transduction were associated with a high number of transgene insertions, while CRISPR-Cas9 was associated with more precise and limited transgene insertion in the edited cell lines. According to the study, OGM identified complex and cryptic structural variants and copy number changes in engineered cells that were not detected by traditional cytogenetic and sequencing-based methods, suggesting potential off target genomic alterations from editing processes.

In plant breeding applications, researchers from UC Davis and UC Berkeley's Innovative Genomics Institute demonstrated that an engineered version of a "jumping gene" enzyme called TnpB could edit the genome of tobacco plants via a one-step process. The method was highly efficient, and the resulting gene edits were inherited by more than 90% of the next generation of plants—a heritability rate that matches that of Cas9.

Gene editing has enormous potential to help feed the world's growing population, but it's currently difficult, time-consuming, and only works in some plant species. A big part of the problem is CRISPR/Cas9's size: it's too large to be delivered into plant cells. Scientists often use viruses to deliver gene-editing machinery to plant and animal cells because viruses naturally insert DNA and RNA into the cells they infect. However, plant viruses have a cargo limitation, and CRISPR/Cas9 is too big for them to deliver.

TnpB is associated with transposons or "jumping genes", short DNA sequences that can move between different parts of the genome by using a similar "cut and paste" mechanism to CRISPR/Cas9. However, TnpB is only around 400 amino acids compared to Cas9's 1300 amino acids—a much more manageable size for viral delivery.

Naturally occurring bacterial TnpB has been shown to edit genes in human and plant cells, but it only works around 3-10% of the time. To improve TnpB's gene editing efficiency, researchers tested two enhanced versions of TnpB (eTnpBc and eTnpBe). To increase heritability, the researchers also added a short RNA sequence that helps the viral spread to the plant's germline, the cells that produce a plant's eggs and sperm.

The researchers tested the engineered TnpBs in tobacco plants (Nicotiana benthamiana) and used tobacco rattle virus as their delivery system. To make the gene edits easily detectable, they disrupted a gene with a visible role: Phytoene desaturase (PDS), which is involved in pigment synthesis. When PDS is switched off, plant tissue turns white.

When they injected two-and-a-half-week-old tobacco seedlings with TnpB-carrying viruses, white spots appeared on the leaves as the virus spread through the plants. The team's molecular analysis confirmed that eTnpBc, which achieved gene editing efficiency of up to 70%, was more efficient than eTnpBe or the naturally occurring version of TnpB, which induced editing efficiencies of 26% and 12%, respectively.

eTnpBc was an even more efficient gene editor when the team tasked it with editing ChlH, a gene that is involved in chlorophyll synthesis. eTnpBc achieved 90% gene editing efficiency for ChlH. To test whether the gene edits were heritable, the researchers collected and sprouted the gene edited plants' seeds. They showed that the gene edits targeting PDS and CHlH were both highly heritable: 89% of the seedlings grown from PDS-edited plants with white pods were completely white.

The increasing complexity of next-generation biologics such as bi- and trispecific antibodies, antibody-drug conjugates (ADCs), and vaccines demands novel, enabling technologies for CHO genome engineering. Recent efforts in the development of hyperactive transposase systems coupled with synthetic biology have created a single, efficient tool for genetic knock-ins, knock-downs, and for complex bioengineering needs.