The Prime Editing System Inserts Complete Genes in Human Cells, A New Approach to Gene Therapy!

Twin prime editing, a CRISPR-based gene editing technique, could be a new and safer approach to gene therapy.

A new version of primary editing has been developed by researchers at the Broad Institute of MIT and Harvard that can install or swap out gene-sized DNA sequences. Prime editing, which was first developed in 2019, is a precise method for performing a wide range of gene edits in human cells, including small substitutions, insertions, and deletions.

The team describes twin prime editing (twinPE), a technique that uses two adjacent prime edits to introduce larger DNA sequences at specific locations in the genome with few unwanted byproducts, in a study published in Nature Biotechnology on December 9, 2021. With further development, the technology could be used as a new type of gene therapy to insert therapeutic genes into mutated or missing genes in a safe and highly targeted manner.

The researchers used twinPE to edit a gene linked to Hunter syndrome, a rare genetic disorder, in human cells, demonstrating its therapeutic potential. An inversion of a specific 40,000 base pair long stretch of DNA causes this disease. The researchers utilised twinPE to introduce a similar-length inversion at the same location in the genome, demonstrating how the technology could be employed to fix the disease-causing mutation. The researchers also employed twin PE to precisely insert thousands of base pairs of gene-sized DNA cargo into therapeutically important places in the genome.

The method circumvents a flaw in the original prime editing mechanism, which can only edit a few hundred base pairs. However, some genetic diseases may necessitate more extensive editing in order to study or treat them. TwinPE, like the original prime editing approach, does not entirely split the DNA double helix by cutting both strands at the same time, which can result in erratic editing and dangerous chromosomal abnormalities.

“One of the longstanding challenges in gene editing has been inserting a healthy gene in a patient at a site of our choosing without generating double-strand breaks and mixtures of byproducts,” said David Liu, senior author of the study, Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute, professor at Harvard University, and a Howard Hughes Medical Institute investigator.

“TwinPE could be a potentially safer and more precise way to insert whole genes of therapeutic interest into positions we specify, such as the location of the native gene in healthy individuals or ‘safe harbor’ sites thought to minimize the risk of side-effects.”

Editing during peak hours

Liu’s lab developed Prime editing, which allows for DNA substitutions, insertions, and deletions, and promises to fix the bulk of disease-causing genetic variants. Prime editing technology has recently improved in efficiency, bringing it closer to therapeutic uses. Sequences longer than 100 base pairs, on the other hand, remained inefficient to edit.

This void is filled by twin prime editing. A prime editor protein and two prime editing guide RNAs are used in the system to guide the editing machinery and encode edits. Each of the two guide RNAs instructs the editing protein to form a single-stranded nick in the DNA at distinct targeted spots throughout the genome, avoiding the type of double-strand break that can result in undesirable byproducts in other approaches. After that, the system creates two new complementary DNA strands with the required sequence in the space between the two nicks. The team was able to insert, substitute, or delete sequences up to 800 base pairs long using this method.

The researchers used their twin prime editing approach to create “landing spots” in the genome for enzymes known as site-specific recombinases, which catalyse the integration of DNA at specified locations in the genome. The team then used a recombinase enzyme to treat the cells before inserting the long pieces of DNA into the genome. The scientists were able to edit sequences that were thousands of base pairs long — the length of entire genes — by combining twinPE and recombinase enzymes.

Liu and his colleagues are now experimenting with alternative recombinases to see if they can improve twinPE’s efficiency. They’re also looking at how well twinPE can install much longer genetic sequences.

“It’s been a longstanding aspiration of many labs including ours to be able to advance gene therapy in the way that scientists have advanced gene editing over the past several years,” Liu said. “This study, together with other efforts of other scientists, could mark the beginnings of a new generation of gene therapy strategies, just as CRISPR nucleases, base editors, and prime editors represented the beginnings of a new generation of gene editing technologies.”

Andrew V. Anzalone, Xin D. Gao, Christopher J. Podracky, Andrew T. Nelson, Luke W. Koblan, Aditya Raguram, Jonathan M. Levy, Jaron A. M. Mercer, and David R. Liu, “Programmable deletion, replacement, integration, and inversion of large DNA sequences with twin prime editing,” Nature Biotechnology, 9 December 2021.

The Merkin Institute of Transformative Technologies in Healthcare, the National Institutes of Health, and the Howard Hughes Medical Institute all contributed to this research.

Written by IOI

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