With Two RNA Guides, a new gene Editing Technique allows Long DNA Insertions

Good news! Low efficiency still seems to be an issue. More progress to be expected.

"... Traditional prime editing uses a single pegRNA to deliver a Cas9 enzyme to the targeted DNA sequence. The Cas9 then nicks one strand to make room for an insertion. The pegRNA serves as a reverse transcription template for a single DNA strand that’s integrated into the genome and whose opposing strand is written by the host cell’s DNA repair machinery. By providing a second pegRNA-Cas9 complex with a partially complementary sequence to the first, the GRAND system generates a double-stranded structure that is less likely to be digested by cellular enzymes and has higher odds of inserting into the genome. With this system, the researchers inserted 150-base-pair-long stretches with 63 percent efficiency, 250-base-pair insertions with 28.4 percent efficiency, and 1,000-base-pair segments with less than one percent efficiency. ..."

From the abstract:

"Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 base pairs) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences."

With Two RNA Guides, Editing Technique Writes Long DNA Insertions | TS Digest | The Scientist The approach, called GRAND, uses a second guide RNA to write complementary strands of DNA in targeted genomic locations, resulting in efficient insertions that can be hundreds of base pairs long.

Efficient targeted insertion of large DNA fragments without DNA donors (no public access)