The development of CRISPR/Cas genome editing techniques has been one of the most exciting recent events in the field of genome editing. Because of its relative ease of use researchers have begun using CRISPR/Cas for a wide range of applications including gene tagging, knock-out of a target gene, and introduction of a specific mutation, insertion, or deletion. It has been used to create disease models, study gene function, improve agriculture, and more with even further applications expected in the future.
Previous genome editing methods, such as zinc-finger nucleases (ZFNs) and transcription activator-like effect nucleases (TALENs), perform similar functions to the CRISPR/Cas system, but with a few notable differences. The CRISPR/Cas system does not require protein engineering for each target sequence and is thus much easier to design, has a higher efficiency, and allows for multiplexing (simultaneous creation of multiple mutations in one genome). The relative simplicity and flexibility of CRISPR/Cas technology have contributed to its widespread use.
CRISPR-based genome editing requires two key components, a guide RNA (gRNA) that compliments the targeted DNA and the Cas9 nuclease that cuts the DNA at the correct site. The guide RNA is composed of two sequences – a CRISPR RNA (crRNA) that guides the gRNA:Cas9 complex to the correct DNA sequence in the target cell and a trans activating crisper RNA (tracer RNA) that is complementary to the Cas9 nuclease. The function of the Cas9 endonuclease is to cleave DNA if there is a sufficient match between the gRNA and the target sequence in the cell.
The process of genome editing using CRISPR-Cas9 will differ based on intended results, the cell types being worked on, and other variables, but the general steps involve:
Selecting a target cell type and designing a gRNA
- Select a target that is unique and upstream of a Protospacer Adjacent Motif (PAM), a short DNA sequence that helps guide Cas9 to the correct sequence but is not itself cleaved. The PAM will vary based on the target cell type.
- Design your gRNA or choose a validated gRNA. Factors to consider include: GC content of the target DNA, length of the target DNA, and avoidance of potential off-target effects. Off-target effects occur when there are similarities between the target sequence and other downstream sequences which can cause cleavage at unintended sites. Several free tools are available to help researchers find a target sequence with minimal amounts of off-target effects such as CRISPR Design from the Zhang Lab at MIT and CHOPCHOP from Harvard.
- Synthesize and clone gRNA.
A general overview of CRISPR/Cas genome editing: gRNA and Cas9 are delivered into the cell in order to create a mutation, deletion or insertion in the target sequence.Selecting a Cas9
- There are several Cas9 variants based on species, but the most common is from S. pyrogens. The type will depend on the PAM sequence you select.
- Cas9 will cleave the target if there is enough similarity between the target sequence and gRNA.
Delivering Cas9 and gRNA into the target cell
- Decide how the selected Cas9 and gRNA will be delivered into the cell. The most common method is to insert them into a plasmid prior to transfection into the cell, but they can also be inserted into a viral vector or the components can be directly delivered into the cell.
- The method of insertion into the target cell will depend on the specifics of the experiment.
- Physical transfection, such as electroporation, is generally the cheapest option but has lower efficiency which makes it ineffective for hard to transfect cell types.
- Chemical methods, such as lipid mediated transfection, can have variable efficiency and while they are sometimes a cheap and effective option, some chemical transfection methods are toxic to cells.
- Viral transduction has relatively high overall efficiency, but can be difficult to produce and raises concerns when being considered for human use because of its potential to trigger immune reactions or cause toxicity in cells.
Analysis of results
Analysis via electrophoresis, real time PCR, next-generation sequencing, or flow cytometry of finished product compared to control cells should reveal genomic differences between the two. One common assay to verify results is the mismatch-cleavage assay. It consists of PCR amplification and analysis via electrophoresis. All-inclusive kits to perform this and other assays are available to purchase from numerous companies.