New CRISPR-based tool for find-and-replace editing of DNA

Ygggdrasil

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Summary
Scientists have developed an improved CRISPR-based gene editing tool that can edit DNA more efficiently and flexibly than existing tools.
Over the past half decade, researchers have envisioned the CRISPR-Cas9 genome editing system as a way to correct pathogenic mutations in patients, but these reserachers face a number of hurdles in developing the technology for clinical applications. While CRISPR is very efficient at cutting DNA to introduce mutations that can knockout gene function, it is much less efficient at introducing specific changes in the DNA sequence, such as those that would correct the mutations that lead to genetic diseases like sickle cell disease. Very often, the traditional CRISPR approach would produce many cells with unwanted mutations and only a few containing the desired DNA sequence change. This is obviously a problem if one wants to use this approach to correct mutations in a patient's body.

A few year ago, researchers developed new versions of CRISPR that could introduce specific base changes without cutting DNA. These base editors promised to allow researchers to make specific changes to DNA more efficiently and with fewer unwanted changes (though, it was later found that some versions of these base editors did lead to quite a number of unwanted mutations). One important limitation of these base editors, however, is that they were capable only of a single type of DNA base change, such as a converting an A to a G or a C to a T, and base editors for all of the possible changes have not been developed (indeed, it took quite an effort to develop a base editor capable of changing A to G, and the remaining base changes would likely be even harder to develop base editors for due to the underlying chemistry of the nucleobases).

Today, however, a team of researchers lead by chemist David Liu at the Broad Institute reports the development of an improved CRISPR system they call a "prime editor:"

Today, in the latest—and possibly most important—of recent improvements to CRISPR technology, Liu is introducing “prime editing,” a molecular gadget he says can rewrite any type of genetic error without actually severing the DNA strand, as CRISPR does.

The new technology uses an engineered protein that, according to a report by Liu and 10 others today in the journal Nature, can transform any single DNA letter into any other, as well as add or delete longer stretches. In fact, Liu claims it’s capable of repairing any of the 75,000 known mutations that cause inherited disease in humans.
(source: https://www.technologyreview.com/s/614599/the-newest-gene-editor-radically-improves-on-crispr/)

To allow more flexible editing, the prime editor carries a reverse transcriptase enzyme linked to the Cas9 protein that allows the prime editor to write a short sequence of DNA into the genome at the location specified by the CRISPR component of the prime editor.

Citation to the paper:
Anzalone et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. Published online 21 Oct 2019 https://www.nature.com/articles/s41586-019-1711-4

Abstract:
Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2–5. 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.
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David R Liu, BTW, isone of the patent-holders for Crispr, which is held by Editas (EDIT), the company he founded. wonder if this potentially gives EDIT an advantage over the other two Crispr companies, CRSP and NTLA
 

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