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Prime Assembly enables kilobase‑scale DNA insertion for CAR‑T cell engineering Prime Assembly is a variant of prime editing that uses RNA guides to direct the insertion of donor DNA fragments thousands of bases long. It was described in a study published August 28 in Nature Biotechnology. The method creates complementary 3′‑overhangs on the genome and donor DNA; when these ends anneal, the donor sequence is filled in at the junction. The authors first tested the approach in the HEK293T cell line with donor fragments ranging from 1.0 to 6.5 kilobases. Using a 2.9‑kb donor, the replacement frequency reached 57.8%, and long‑read DNA sequencing showed that most inserted fragments retained the correct sequence. They then applied the same scheme to primary human T cells, inserting a chimeric antigen receptor (CAR) that recognizes CD19 on B‑cell malignancies. With two pairs of overhangs, CAR was detected on the surface of 28.1% of cells after seven days. The resulting CAR‑T cells killed CD19‑positive tumor cells in culture and suppressed tumor growth in immunocompromised mice, extending survival relative to controls. In a final experiment, the researchers delivered plasmid DNA to mouse livers via tail‑vein injection. Among hepatocytes that took up the plasmids and fluoresced green, correct genomic insertion was achieved for 4.3% of cells with one Prime Assembly variant. Thus, both replacing a DNA segment and assembling a CAR‑T cell rely on the same principle: ligating donor DNA to a chosen genomic site via complementary overhangs. The HEK293T line tests the ligation itself, primary T cells assess the functional product, and mouse liver demonstrates the feasibility of the edit in living tissue. 🔗 Read original →