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Researchers in China have developed a new method that allows them to insert long stretches of DNA into a genome. The process, named GRAND editing, is one of a handful of new systems aimed at using two partially complementary prime editing guide RNAs (pegRNAs) to increase the efficiency of large-scale insertions, says David Liu, a Howard Hughes Medical Institute investigator and gene editing researcher at the Broad Institute of MIT and Harvard University who was not involved in the new study. Thanks to the stability of the double-stranded DNA that forms when those pegRNAs are reverse transcribed, these tools up the chances that the newly written DNA will be integrated into the genome in place of the native sequence.

Hao Yin of the Medical Research Institute of Wuhan University tells The Scientist that he was motivated to develop the technique because existing gene editing platforms are “highly effective for gene knockout but not targeted insertion.” Yin says it was important to develop a way to insert large fragments into a genome because of the therapeutic potential of such an approach for genetic conditions.

Yin explains that with conventional prime editing, insertions of just a few dozen base pairs are made with only about 20 percent efficiency, and a preprint published last year shows that prime editing is basically nonfunctional for insertions of more than 66 base pairs, which is considerably shorter than the typical human indel. GRAND editing can consistently insert sequences of a couple hundred base pairs, and could occasionally create insertions of 1,000 base pairs, according to a study Yin and colleagues published in Nature Methods in February. 

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. 

Infographic of two prime editing guide RNAs (pegRNAs), deliver Cas9 enzymes to targeted regions of the genome.
Two prime editing guide RNAs (pegRNAs), deliver Cas9 enzymes to targeted regions of the genome. The enzymes nick opposite strands of the DNA at different points, at which point the pegRNA serves as a template for the desired gene insertion. This results in two single-stranded DNAs (ssDNAs) that bind to each other due to their partially complementary sequence and, if they outcompete the original DNA, become integrated into the genome and completed by the host cell’s existing DNA repair machinery. Because two pegRNAs are used, GRAND editing can insert segments of up to 1,000 base pairs long—more than what one pegRNA could deliver alone. WEB | PDF

Liu, who helped pioneer another new genome-editing method called base editing, tells The Scientist via email that the new study “adds to a growing set of publications” that use two pegRNAs to insert longer segments of DNA. Indeed, Liu coauthored a 2021 Nature Biotechnology paper describing a similar approach that allowed the team to successfully insert a plasmid that was more than 5,000 base pairs in length. “These dual-pegRNA approaches show how creative applications of prime editing can open up exciting new applications, including some that could contribute to new forms of gene replacement therapy,” writes Liu.

Meanwhile, Yin says over email that he and his team are “working on improving the efficiency of GRAND editing [for] 1 kb size sequences” and otherwise optimizing the technique to make it a more robust means of editing genomes.