Precise CRISPR tool (with few, if any, off-targets) could tackle numerous genetic diseases

Despite all the ease with which the popular CRISPR/Cas9 gene-editing tool is able to alter genomes, this method is still somewhat

prone to errors and unintended effects (so-called “off-targets”). To achieve the best outcome, researchers would like to exchange or remove a single nucleotide, without disturbing any other nucleotides among the ~3 billion in the haploid genome. Now, an alternative method offers greater control over genome edits. This alternative method, called “prime editing”, improves researchers’ chances of accomplishing only the edits they want, instead of an unknown number of undesirable off-targets. Authors [see attached article and editorial] suggest that prime editing might help researchers tackle nearly 90% of the more than 75,000 disease-associated DNA variants listed in ClinVar (a database developed by the US National Institutes of Health).

The ability to create virtually any targeted change in the genome of any living cell or organism (animals, plants, fungi, bacteria, even viruses) is a longstanding aspiration of life sciences researchers. Programmable nucleases such as CRISPR/Cas9 generate double-strand DNA breaks (DSBs) that can disrupt genes by inducing mixtures of insertions and deletions at target sites. DSBs, however, are associated with undesired outcomes — including complex mixtures of products, translocations, and even p53 activation. Moreover, the vast majority of pathogenic alleles (remember each gene has one allele from each parent) arise from specific insertions, deletions, or base substitutions that require more precise editing technologies to correct. Homology-directed repair (HDR), stimulated by DSBs, has been widely used to install precise DNA changes.

Prime editing 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. Authors [see attached article] completed more than 175 edits in human cells — including targeted insertions, deletions, and all 12 types of point mutation — without requiring DSBs or donor DNA templates.

Authors applied prime editing in human cells to efficiently correct (with few byproducts) the primary genetic causes of sickle cell disease [requiring a transversion (i.e. changing a pyrimidine to a purine) in the HBB gene] and Tay-Sachs disease (requiring a base-pair deletion in the HEXA gene). Four human cell lines and primary post-mitotic mouse cortical neurons were shown to support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over HDR, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than the Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing. 😊


Nature & editorial, 24 Oct 2019; 574: 464-465

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