All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System
筆記録
Begin by adding all-in-one CRISPR plasmid to immune cells present in a tube. This plasmid contains a Cas9 gene in combination with the sgRNA or single-guide RNA encoding sequence, complementary to the target sequence in the host genome.
Add a linearized donor plasmid containing a transgene sandwiched between homology arms or HAs – the DNA repeats necessary for homologous recombination. Perform electroporation, which uses electric current to inject the plasmid mix into the cell. Once inside, the processed sgRNA forms a complex with the expressed Cas9 nuclease.
The sgRNA binds the target site, allowing the Cas9 nuclease to identify the specific recognition sequence known as protospacer adjacent motif or PAM, and cleave the host DNA near it. This creates a blunt-ended double-strand break where the cellular machinery resects the DNA, generating 3' overhangs. The presence of HA flanking sites triggers the homology-directed repair mechanism where the cut DNA joins the HA sequence, forming a heteroduplex structure – the D-loop.
DNA synthesis continues, forming a strand complementary to the transgene sequence until it rejoins the original DNA strand. The top of the D-loop acts as a template to synthesize the missing part of the DNA, generating the double Holliday junctions – structures where four DNA strands interact. These junctions resolve and the gaps repair, facilitating target gene insertion.
