Sponsored Insight
CRISPR is the coolest technology in a scientist’s toolbox these days because of its unique blend of being extremely powerful yet relatively simple to use. The technique relies on two basic components: a CRISPR-associated (Cas) nuclease that cuts DNA and a guide RNA (gRNA) that tells the nuclease precisely where in the genome to cut. The CRISPR system enables scientists to make changes virtually anywhere in the genome of any organism.
The possibilities of such a technology are vast, from curing genetic diseases to making crops resistant to pests.
Given the immense potential of CRISPR, scientists all across the world are excited to start using it in their research. Once researchers have familiarized themselves with the basics of CRISPR, the next step is to start editing genomes in their own experiments.
However, the path to becoming a genome engineer is not as straightforward as one might think.
For instance, scientists planning to use CRISPR will need to ask themselves the following:
What is the best way to design my gRNA?
Which nuclease is appropriate for my experiment?
Is one transfection method better than the other?
How do I choose the best CRISPR analysis tool?
All these decisions can be overwhelming, and it’s sometimes hard to even know where to start.
Getting Started with CRISPR – The Easy Way
To make all this a bit simpler, let’s consider the entire CRISPR experiment subdivided into three broad steps: Design → Edit → Analyze. In short, scientists must first design their gRNA, then edit the genome, and lastly analyze their results.
This holistic view of the CRISPR experiment is what we call Full Stack Genome Engineering.
Step 1: Design the CRISPR Guide RNA and Select the Cas Nuclease
The first step in your CRISPR experiment is to design a gRNA to target your DNA sequence. The editing efficiency and specificity of the guide RNA will greatly influence the success of your CRISPR experiment. There are different formats and ways of making guide RNAs. The guide RNA format of choice for many of the top genome engineers is synthetic single guide RNAs (sgRNAs) that are chemically modified so that they persist longer within cells.
The design of your gRNA also depends on the nuclease you intend to use, as each gRNA must bind near a nuclease-specific PAM sequence. Although Cas9 is a currently the most popular Cas, a variety of alternative nucleases exist. Therefore, it is worth doing some research on the appropriate CRISPR nuclease depending on the application and specific experimental needs.
Step 2: Edit DNA Precisely with CRISPR
After all CRISPR components have been selected, one more decision remains. What is the best delivery system to introduce the CRISPR components into the cell? There are several options for transfecting cells, all which have different advantages for different cell types.
Once a transfection method is chosen, you must optimize a multitude of conditions in order to ensure that a high amount of editing takes place in your cell type. This can be a laborious process but is critical to achieving high editing efficiency.
Step 3: Analyze Data from the CRISPR Experiment
You picked the best gRNA and used an optimum transfection method, and now you need to analyze your CRISPR edits. Choosing a user-friendly and accurate analysis method is essential to understanding how well the editing worked and to determine the next steps in your experiment. With your editing efficiency and knockout score known, you can now decide whether to perform assays on your pool of cells or perhaps continue to generate clones.
Synthego’s Full Stack Genome Engineering Approach
Now that we have walked through the Design → Edit → Analyze steps of the CRISPR workflow, we hope that you have a better idea of all the work that goes into a CRISPR experiment.
Synthego’s Full Stack Genome Engineering solutions encompass the entirety of CRISPR experiments. We provide comprehensive support for every step of the genome engineering workflow, allowing all scientists to access CRISPR and advance their research.
Our automated, full stack genome engineering platform enables broader access to CRISPR to accelerate basic scientific discovery, uncover cures for diseases and develop novel synthetic biology applications.
Learn more at Synthego.com.