General procedure
- Designing and synthesizing guide RNA (sgRNA): The first step in CRISPR-Cas genome editing is to design and synthesize a small RNA molecule called sgRNA. The sgRNA is designed to specifically target and bind to a specific DNA sequence within the genome, guiding the Cas nuclease to the desired site for editing.
- Delivering CRISPR components into human cells: Once the sgRNA is synthesized, it is typically combined with the Cas nuclease enzyme to form a CRISPR-Cas complex. This complex needs to be delivered into the target human cells. There are several methods for delivering CRISPR components into human cells, including plasmid DNA transfection, mRNA with sgRNA, viral vectors, and ribonucleoprotein (RNP) complexes. The choice of delivery method depends on the specific experimental requirements and the type of human cells being targeted.
- Cas nuclease cleavage of DNA at target site: Once the CRISPR-Cas complex is inside the nucleus of human cells, the sgRNA guides the Cas nuclease to the target DNA sequence, where it cleaves the DNA, creating a double-stranded break (DSB) at the desired site.
- Cellular repair of DSB: The cell's natural DNA repair mechanisms come into play to repair the DSB created by the Cas nuclease. There are two main DNA repair pathways that can be utilized for genome editing: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is an error-prone repair pathway that often results in small insertions or deletions (indels) at the DSB site, leading to gene disruption or knockout. HDR, on the other hand, is a more precise repair pathway that can be used to introduce specific DNA sequences at the DSB site, allowing for precise gene editing or insertion of desired sequences.
- Verification of edited cells: After the CRISPR-Cas genome editing procedure, the edited human cells need to be verified to confirm the desired changes. This is typically done through various molecular techniques, such as PCR (polymerase chain reaction), DNA sequencing, NGS, and/or functional assays, to confirm the presence of the desired edit and assess the efficiency of the editing process.
It's important to note that CRISPR-Cas genome editing in human cells is a rapidly evolving field, and the specific details and protocols may vary depending on the specific application, the type of cells being targeted, and the desired outcome. It is crucial to follow proper experimental protocols, consider ethical implications, and comply with applicable regulations and guidelines when conducting CRISPR-Cas genome editing experiments in human cells.
