The new editor-targeted genome engineering in the absence of homology-directed repair.

Abstract

The CRISPR/Cas9 genome editing technique has become one of the most powerful tools in molecular biology. Discovered in bacteria, the minimal components required for the genome editing activity have quickly been transferred to the mammalian system; a small chimaeric RNA molecule, also called single guide RNA (sgRNA) and the Cas9 endonuclease.1 While the sgRNA guides Cas9 to a specific genomic locus, Cas9 induces DNA double-strand breaks (DSB) with its endonuclease activity. Through a process known as non-homologous end joining (NHEJ), imperfect repair of these DNA breaks in a gene can lead to inactivating frameshift mutations and therefore knockout of the gene.2,3 However, if the DSB occurs in the presence of a DNA donor template with homology to the surrounding DNA sequence, homology-directed repair (HDR) mechanisms can lead to perfect repair of the DNA and incorporation of sequences of interest. These incorporated sequences act as a medium for gene editing and can be up to several kb in length, allowing the potential correction or reversion of a disease-causing mutation. Although the CRISPR/Cas9 HDR system has the potential for clinical therapeutic interventions, there are a number of limitations and caveats with this approach, thereby restricting its widespread use in its current form.