iCRISPR Methods and Tools Shortly after its discovery, the CRISPR/Cas9
system has taken the world of gene editing by storm.
The CRISPR/Cas9 system has become the go-to technique when it comes to mutating and editing
DNA in life science research.
The beauty of CRISPR/Cas9 system is that it only requires two components: a Cas9 enzyme
to snip target DNA and an RNA molecule called the single strand guide RNA (sgRNA) to direct
the Cas9 enzyme to the precise spot through complementary DNA pairing.
In our previous video we introduced the CRISPR/Cas9 system briefly; here we will go over the methods
and tools available to you for using the CRISPR/Cas9 system in practice.
We invite you to watch our previous video first before continuing with this one.
Genome editing is achieved once the two components of the CRISPR/Cas9 system have been expressed
in living cells.
The delivery efficiency of the Cas9 endonuclease along with the gRNA directly affects the successfulness
of genome editing.
There are several methods to express these components in living cells, either though
non-viral or viral gene delivery systems.
There are two common non-viral gene delivery procedures available: 1.
chemical methods such as lipid-mediated transfection or calcium phosphate transfection, 2.
physical methods including electroporation or laser-mediated transfection.
Lipid mediated transfection involves the use of cationic lipids or cationic polymers.
First, the negatively charged phosphates of the DNA backbone, associate with these positively
charged lipid or polymers forming a complex called liposome.
The liposomes, which are essentially vesicles containing the exogenous DNA, can then merge
with the cell membrane and release their content inside the cytoplasm.
The advantage of applying lipid mediated transfection is its high efficiency in a broad range of
cell lines; however, that efficiency depends on the optimization of culture conditions
for each cell type.
The principle of calcium phosphate transfection involves mixing the DNA with calcium phosphate
to ‘condense’ the DNA and form a calcium-phosphate-DNA-co-precipitate.
The calcium phosphate also helps in binding to the cell surface, and from there, the DNA
enters the cell by endocytosis.
Calcium phosphate transfection has been a common method since the early 1970s due to
its inexpensive components that are readily available.
Depending on the cell type, calcium phosphate may not be a suitable transfection method
as it can have cytotoxic effects.
In addition, reagent consistency, such as small changes in pH, can result in compromised
transfection efficiency.
Electroporation, as the name suggests, uses an electrical pulse to physically create temporary
pores in cell membranes to allow foreign DNA to enter the cells.
This method allows various types of DNA to be introduced readily into cells without limitation
of DNA size.
On the other hand, the main concern with this method is the substantial cell death caused
by the high voltage pulses.
The use of this method requires more cell numbers compared to chemical transfection
methods.
While the non-viral methods mentioned are generally sufficient to deliver foreign genes
into cells, most of the time they are transient expressions and the efficiency can be low
depending on the cell type.
In order to maintain gene expression with high gene delivery efficiency, the viruses
are still the preferred system for many researchers.
There are three types of viruses commonly used for gene delivery in living cells.
These include lentivirus, adenovirus, and adeno-associated virus.
All three types of viruses required to be modified into replication deficit viruses
in order to be applicable for gene delivery.
Lentiviruses are single-stranded RNA viruses belonging to the retroviridae family.
Lentiviruses enter the cells through the interaction of viral envelope protein and cell surface
receptors.
Once inside, they will stably integrate their genome to host-cell chromosomes.
Long term transgene expression is thus achieved in target cells which provides a major advantage
to gene expression researches.
Adenoviruses are well-characterized, non-integrating linear dsDNA viruses that enter target cells
via receptor-mediated endocytosis.
They are known for their outstanding delivery capacity of foreign DNA fragments up to 36
kb, as well as their ability to give almost 100% transduction rates on many dividing and
non-dividing cell types.
Instead of combining its own DNA with the genome of the host cell, adenoviruses remain
as an episome within the infected cells giving transient gene expression.
One important thing to note about adenoviruses is that they tend to cause strong immune and
inflammatory responses among animal models.
Amongst all recombinant viruses, Adeno-associated virus (AAV) is the most promising candidate
for gene therapy owing to its 2 properties: non-integrating nature and the lack of pathogenicity
in animal models.
AAV is also a very attractive tool in regenerative medicine due to its differential tropism that
can be achieved with difference AAV serotypes.
For instance, AAV2 presents natural tropism only towards skeletal muscles, neurons and
hepatocytes.
The downside of AAV application is its naturally small genome size restricting its cloning
capacity.
The viruses offer a very simple procedure to gene delivery allowing for high efficiency
transduction in both proliferating and quiescent cells.
These viruses are often technically challenging and labor intensive to produce.
Efficient delivery of the Cas9 and gRNA into a living cell is required to carry out genome
editing.
abm features cas9 expression tools in the form of plasmid vectors, proteins, lentiviruses,
adenoviruses and AAVs.
In addition to the Wilde Type cas9 nuclease, abm also provide the cas9 nickase in all formats,
so depending on your application, you can choose the best expression system for your
research project.
abm also offers genome-wide sgRNA library ready for you to use.
We provide individual lentivirus constructs or in a set of 3, which will provide you 3
different sgRNA designs targeting different locations of your target gene.
They can be used separately or pooled together to achieve optimal gene knockout for your
system.
Similarly, our iCRISPR sgRNA adenoviruses and AAVs are useful for non-integrating, highly
efficient genome editing in hard-to-transduce target cells.
Although Cas9 and gRNA can be expressed from separate plasmids or viruses, researchers
also have the option of using a single construct that expresses both Cas9 and gRNA.
This could be helpful when working with animals or immune cells that are not very susceptible
to gene delivery.
Abm offers an All-in one system with dual expression of cas9 and gRNA in a single lentivector/lentivirus.
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