Gene editing with clustered regularly interspersed short palindromic repeats (CRISPRs)/Cas9.
In bacteria, DNA sequences consisting of CRISPRs are transcribed into guide RNAs (gRNAs) with a constant region and a variable sequence of about 20 bases.
The constant regions of gRNAs bind to Cas9, permitting the variable regions to form heteroduplexes with homologous host cell DNA sequences.
The Cas9 nuclease then cleaves the bound DNA, producing a double-stranded DNA break.
To perform gene editing, gRNAs are designed with variable regions that are homologous to a target DNA sequence of interest.
Coexpression of the gRNA and Cas9 in cells leads to efficient cleavage of the target sequence.
In the absence of homologous DNA, the broken DNA is repaired by non homologous end joining (NHEJ), an error-prone method that often introduces disruptive insertions or deletions (indels).
By contrast, in the presence of a homologous “donor” DNA spanning the region targeted by CRISPR/Cas9, cells instead may use homologous DNA recombination (HDR) to repair the DNA break. HDR is less efficient than NHEJ, but has the capacity to introduce precise changes in DNA sequence.
Potential applications of CRISPR/Cas9 coupled with HDR include the repair of inherited genetic defects and the creation of pathogenic mutations.