144 DNA landing site variants improved the insertion of large genetic cassettes in human cells and rice
144 DNA landing site variants improved the insertion of large genetic cassettes in human cells and rice
In a paper published on 2 September, the authors modified the short DNA landing site recognized by the bacteriophage enzyme Bxb1. The best variants increased the frequency of successful integration in the human HEK293T cell line. In rice, the same approach, combined with an engineered Bxb1, enabled the insertion of a long genetic cassette at a selected genomic site.
Some genetic engineering tasks require inserting an entire gene together with the sequences that regulate its activity. This cargo must be integrated at a predetermined genomic site. In the PASSIGE method, a prime editor first installs a short attP landing site at that location. Bxb1, an enzyme derived from a bacteriophage, then recognizes attP and its partner site, attB, on the DNA cargo and joins them.
In earlier work, the researchers made Bxb1 more efficient by modifying its protein sequence. This time, they tested changes to the landing site that the enzyme recognizes on the cargo. They combined three sequence segments found in natural att sites to produce 144 attB variants. In HEK293T cells, the three best variants achieved 49–52% recombination among alleles in which the prime editor had already installed attP. On average, this was 1.7 times the rate obtained with the original attB.
Efficient, precise insertion of large DNA sequences can be improved by engineering both the enzyme and the DNA landing site it recognizes.
The authors tested this combination with larger cargoes. In HEK293T cells, attB(V111) combined with an engineered Bxb1 produced an overall insertion rate of 31% for the CD19-CAR cassette, which encodes a receptor that allows T cells to recognize the CD19 protein, and 25% for the OTC gene. In rice, another engineered version of Bxb1 combined with V111 precisely inserted a sequence 5,8 thousand nucleotides long in 31 of 129 regenerated transgene-positive plants.
The authors used Sanger sequencing to read the cassette junctions in HEK293T cells. In two rice lines, long-read DNA sequencing confirmed that the complete cassette was present at the expected site, and the insertion was inherited by the plants’ offspring. The raw sequencing reads are available in BioProject.
The overall efficiency of PASSIGE still depends first on how reliably the prime editor installs attP. The new result identifies another variable that affects the system: the sequence of the landing site recognized by Bxb1.