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.