KNIT inserts DNA fragments longer than 10,000 base pairs through a single nick

KNIT inserts DNA fragments longer than 10,000 base pairs through a single nick

On July 22, a paper in Nature described KNIT, a system for the precise insertion of long DNA fragments. In cell experiments, it worked with sequences ranging from 700 base pairs to fragments longer than 10,000 base pairs, and in some tests, up to 89% of the insertions were correct. The authors also inserted a CAR receptor into human T-cells.

Correcting a single letter in DNA and adding a new gene to a cell are different tasks. The second requires inserting a complete genetic instruction into a chromosome, such as a functional copy of a gene or the sequence for a receptor that directs an immune cell toward a tumor. The longer this instruction is, the harder it becomes to insert it at a specific site.

Standard CRISPR-Cas9 cuts both DNA strands. The cell repairs the break while researchers try to incorporate a donor sequence at that site. In KNIT, a Cas9 variant nicks one strand, while an attached protein holds the donor DNA nearby. The ends of the donor DNA match the sequences surrounding the selected chromosomal site.

Additional experiments link this process to DNA repair based on matching sequences: insertion occurred less often when the RAD51 and BRCA2 proteins were suppressed. The single nick specifies the target site, while the donor DNA provides the cell with a template for insertion at that site.

In the authors’ measurements, KNIT produced fewer small insertions and deletions, rearrangements between chromosomes, and changes outside the selected region than editing based on a double-strand break. A 2018 study had already reported large deletions and complex rearrangements following CRISPR-Cas9 cleavage in mouse and human cells. A method that inserts a long fragment without such a break therefore changes the engineering problem itself.

The authors tested whether the inserted sequences functioned. In engineered human cells carrying a LIPA mutation, they added a functional copy of the gene and restored its expression. They then inserted a CAR into human T-cells. These CAR-T-cells recognized and killed tumor cells in culture and in mice.

An immune-cell receptor and a functional copy of a gene require much more DNA than a single-base substitution. KNIT combines a large genetic cargo, a specified target site, and a single nick in one process.