Prime Genome Editing System Could Accelerate Disease Cures

A new type of gene editing enables more precise editing than CRISPR. Prime editing combines two key proteins and a new RNA to make targeted insertions, deletions, and all possible single-letter changes in the DNA of human cells.

Prime Editing is capable of directly editing human cells in a precise, efficient, and highly versatile fashion. The approach expands the scope of gene editing for biological and therapeutics research, and has the potential to correct up to 89 percent of known disease-causing genetic variations.

* More cells and more gene-editing situations will be addressable
* 89 percent of genetic diseases will be treatable with this new editing system
* more edits with fewer errors are possible
* They applied 175 edits without errors in the initial research

Nature – Search-and-replace genome editing without double-strand breaks or donor DNA

Abstract

Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutation without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct about 89% of known pathogenic human genetic variants.

A New CRISPR Approach

Prime editing differs from previous genome-editing systems in that it uses RNA to direct the insertion of new DNA sequences in human cells.

The first CRISPR tool harnessed for genome editing in human cells, pioneered at the Broad Institute, MIT, and Harvard, was the Cas9 protein. Cas9 makes nearby breaks on each DNA strand, cutting the DNA entirely. These tools can disrupt target genes at a specific location and then make it possible to add new sequences through recombination of new DNA into the site, directed by the cell itself.

Base editors, first developed by Liu’s laboratory, build on this technology, fusing Cas9 to proteins that can perform chemical reactions to change a single letter of DNA into another. Current base editors can make four types of single-base changes efficiently: C-to-T, T-to-C, A-to-G, and G-to-A.

The new prime editing system involves coupling Cas9 to a different protein called reverse transcriptase. The molecular complex uses one strand of the target DNA site to “prime,” or initiate, the direct writing of edited genetic information into the genome.

A new type of engineered guide RNA, called a pegRNA, directs the prime editor to its target site, where a modified Cas9 cuts one strand of the DNA. The pegRNA also contains additional RNA nucleotides encoding the new edited sequence. To transfer this information, the reverse transcriptase element reads the RNA extension and writes the corresponding DNA nucleotides into the target spot.

Made to Order Gene Editing

In the Nature paper, the team demonstrated prime editing’s ability to precisely correct gene variants that cause sickle-cell anemia, requiring the conversion of a specific T to an A, and Tay Sachs disease, requiring the removal of four DNA letters at a precise location in the genome.

The researchers further report a variety of successful edit types in human cells and primary mouse neurons, including all 12 possible ways to replace one DNA letter with another, insertions of new DNA segments up to 44 DNA letters long, precise deletions of up to 80 DNA letters, and modifications combining these different types of edits.

“With prime editing, we can now directly correct the sickle-cell anemia mutation back to the normal sequence and remove the four extra DNA bases that cause Tay Sachs disease, without cutting DNA entirely or needing DNA templates,” says Liu, who is also a professor of chemistry and chemical biology at Harvard University and a Howard Hughes Medical Institute investigator. “The beauty of this system is that there are few restrictions on the edited sequence. Since the added nucleotides are specified by the pegRNA, they can be sequences that differ from the original strand by only one letter, that have additional or fewer letters, or that are various combinations of these changes.”

“The versatility of prime editing quickly became apparent as we developed this technology,” recalls Anzalone. “The fact that we could directly copy new genetic information into a target site was a revelation. We were really excited.”

When making precision changes, the researchers report that prime editing achieves successful edits with a lower rate of undesired “off-target” changes when compared to approaches that require making nearby breaks on each DNA strand. Prime editing can also make precise single-nucleotide changes in target sequences that base editors have difficulty accessing, according to the team’s data.

Liu’s team intends to continue optimizing prime editing, including by maximizing its efficiency in many different cell types, further investigating potential effects of prime editing on cells, additional testing in cell and animal models of disease, and exploring delivery mechanisms in animals to provide a potential path for human therapeutic applications.

The researchers and the Broad Institute are making this technology freely available to the academic and non-profit communities, including by sharing vectors through the non-profit Addgene. For these groups, no license is necessary.

The Broad Institute is making prime editing tools available to license non-exclusively for research and manufacturing by companies, and for the commercial development of tools and reagents. For human therapeutic use, Broad Institute has licensed the technology to Prime Medicine under the inclusive innovation model. As has been publicly reported, Beam Therapeutics has received a sublicense from Prime Medicine for the use of prime editing in certain fields and for certain applications. (Liu is a consultant and co-founder of both Prime Medicine and Beam Therapeutics.)

SOURCES Broad Institute, Nature
Written By Brian Wang, Nextbigfuture.com