Since the discovery of the genome-editing tool CRISPR/Cas9, scientists have been looking to utilize the technology to make a significant impact on correcting genetic diseases. Technical challenges have made it difficult to use this method to correct disorders that are caused by single-nucleotide mutations, such as cystic fibrosis, sickle cell anemia, Huntington's disease, and phenylketonuria. However now, researchers from the Center for Genome Engineering, within the Institute for Basic Science (IBS) in Korea, have just used a variation of CRISPR/Cas9 to produce mice with single-nucleotide differences. The findings from this new study were published recently in Nature Biotechnology in an article entitled Highly Efficient RNA-Guided Base Editing in Mouse Embryos.

Although genome editing with programmable nucleases such as CRISPRCas9 or Cpf1 systems holds promise for gene correction to repair genetic defects that cause genetic diseases, it is technically challenging to induce single-nucleotide substitutions in a targeted manner, the authors wrote. This is because most DNA double-strand breaks (DSBs) produced by programmable nucleases are repaired by error-prone non-homologous end-joining (NHEJ) rather than homologous recombination (HR) using a template donor DNA. As a result, insertion/deletions (indels) are obtained much more frequently at a nuclease target site than are single-nucleotide substitutions.

The most frequently used CRISPR/Cas9 technique works by cutting around the faulty nucleotide in both strands of the DNA and cuts out a small part of DNA. In the current study, the investigators used a variation of the Cas9 protein (nickase Cas9, or nCas9) fused with an enzyme called cytidine deaminase, which can substitute one nucleotide into anothergenerating single-nucleotide substitutions without DNA deletions.

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An Efficient Single-Nucleotide-Editing CRISPR - Genetic Engineering & Biotechnology News