New Gene-Editing Platform PRIME-In Inserts Large DNA Into T Cells Without Cutting Both Strands

A Safer Way to Edit Cells

A team of researchers has unveiled a new genome-editing platform that can insert large DNA sequences into human T cells without cutting both strands of DNA, addressing one of the most persistent safety concerns in engineered cell therapies. The platform, called PRIME-In, short for Prime Editing-Mediated Large Integration, was published on 30 April in Nature Biomedical Engineering.

How PRIME-In Works

Unlike conventional approaches that rely on double-strand DNA breaks to trigger the cell's repair machinery, PRIME-In uses a prime editor to nick only one strand of the target DNA. At the same time, it primes a donor plasmid with a short microhomology sequence through reverse transcription. The primed sequence then hybridises to the genomic nick and recruits the cell's own DNA polymerases to extend directly from the donor, sidestepping the need for breaks, recombinases, or two-step landing pad insertions.

An advanced version, PRIME-In 2.0, introduces a second genomic nick via an additional guide RNA, achieving up to 88% knock-in efficiency in HEK293T cells and handling payloads as large as 9.2 kilobases at over 80% efficiency. In primary human T cells, historically among the most difficult targets for non-viral editing, the platform achieved roughly 50% integration efficiency for a 3-kilobase CD19 CAR construct, with an 11-fold T cell expansion in just seven days.

A Striking Safety Profile

The safety data is what really sets PRIME-In apart. Chromosomal translocations occurred at a rate of just 0.2 to 0.4%, compared with 2.34% for homology-mediated end joining, a commonly used alternative. Off-target knock-in events registered below 4% genome-wide, versus 23% for the same comparator. In mouse xenograft models, PRIME-In-engineered CAR T cells cleared tumours at levels comparable to those produced with lentiviral vectors, the current clinical standard.

Implications for Cell Therapy

The platform addresses several bottlenecks that have slowed the transition from viral to non-viral CAR T cell manufacturing. Viral vectors carry risks including insertional mutagenesis and are expensive to produce at scale. Methods that rely on double-strand breaks can cause cytotoxicity and chromosomal abnormalities, complicating regulatory approval. PRIME-In eliminates these concerns whilst maintaining therapeutic-grade efficiency, positioning it as a strong candidate for clinical translation, and arrives amid rapid advances in prime editing more broadly.