eBook: Optimizing Gene-Editing Workflows - 4

Tools and Strategies to Help Ensure
CRISPR Experimental Success
No matter the approach or application, some basic steps should
be followed.
Genome editing is a powerful technique that is
having a tremendous impact across many areas of
biological research and model systems. The simple-to-use
CRISPR-Cas9 system especially has seen
remarkable adoption, from forward- and reverse genetic
screening to gene therapy. Variations on the
system now allow researchers to direct a variety of
tools to specified genomic loci, to affect a host of
applications from epigenomic alteration to imaging.
Yet despite its simplicity, ensuring a CRISPR experiment
is as efficient as possible requires effort in the
planning, execution, and analysis. The articles in this
eBook examine some tools and strategies that help
optimize gene-editing workflows and maximize the
likelihood of experimental success.
What is CRISPR?
Earlier techniques such as TALENs and zinc-finger
nucleases (ZFNs) use an engineered nuclease to
cut the genome at a specified location, with each
nuclease designed for that specific locus. What
the CRISPR-Cas system brings to the toolbox is the
ability of a specific guide RNA (sgRNA) to direct a
generic Cas nuclease to a locus of interest. sgRNA
is far easier, faster, and less expensive to engineer
and produce than ZFN or TALEN proteins. Unique
sgRNAs can be pooled or placed into array formats
to use for screening.
DNA cut by Cas will generally result in repair by endogenous
processes (non-homologous end joining,
NHEJ), either accurately or more likely with
insertions or deletions, that may render the gene
non-functional (create a knockout). If a donor template
with homology to the sequence surrounding
the break is available, it may be inserted into the
break by the endogenous homology-directed repair
(HDR) process in a small percentage of cells. As these
are probabilistic occurrences, it is necessary to determine
what actually has occurred.
Choices
In planning a CRISPR experiment, as most experiments,
there are choices to be made.
Among these is how components are to be delivered.
For example, Cas-expressing cell lines are
readily available, giving the experiment a head start.
The nuclease can also be introduced to the cells as
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eBook: Optimizing Gene-Editing Workflows

Table of Contents for the Digital Edition of eBook: Optimizing Gene-Editing Workflows
