CRISPR and the CRISPR associated protein (Cas) is a powerful genome engineering technology capable of curing genetic disease, feeding more people, improving space exploration, and even helping catch criminals. Simply put, there’s a reason the scientists behind this technology won the Nobel Prize in Chemistry in 2020.
CRISPR was originally identified and characterized in bacteria where it acts as an RNA-based defense mechanism protecting the bacteria from invading bacteriophages (viruses that infect bacteria) (Jinek et al). Thus, in the natural world, CRISPR acts as bacteria’s adaptive immune system.
In 2013, CRISPR/Cas9 was used for the first time for gene editing to create targeted double-stranded DNA breaks, which has since revolutionized molecular biology procedures. And as work continues, the CRISPR “toolbox” continually expands offering scientists the ability not only to break genes (knockout), but also to make precise corrections, additions, deletions, as well as modulate gene expression (epigenetics) and even image the location of genes within the nucleus!
This guide will prepare you for CRISPR experimentation by covering the basics of CRISPR design for knockout experiments. To learn more and practice these techniques, register for CRISPR Classroom’s Gene Knockout: gRNA Design to Analysis online and hands-on mini course.
The Basics of Cas9 complex explained
CRISPR systems fall into three categories – type I, II and III (Makarova et al). Popular CRISPR genome editing tools are adapted and simplified from endogenous type II systems and have the following components:
Cas9: An endonuclease that induces a double-strand break in genomic DNA.
Guide RNA (gRNA): This RNA has a standard scaffold sequence, which is required to bind with the Cas9 protein, and a 20-nucleotide “spacer” sequence defined by the researcher.
How does CRISPR gene knockout work?
When the guide RNA (gRNA) and Cas9 are expressed together in a cell, a gRNA:Cas9 complex forms. This complex has the capability to scan DNA in search for the genomic DNA that corresponds to the 20 bp user-defined spacer sequence fo the gRNA. In addition, the Cas9 is scanning for a specific nucleotide motif called a protospacer adjacent motif (PAM) which is required for complex activation and DNA cutting. The most common PAM motif targeted by commercial Cas9 enzymes is NGG (two guanines followed by any nucleotide).
Upon PAM recognition and gRNA hybridization, the Cas9 enzyme is stimulated to create a DNA double stranded break. This DNA lesion initiates DNA repair processes that eventually lead to the functional outcome: the gene edit.
The most prevalent DNA repair pathway in mammalian cells is Non Homologous End Joining (NHEJ). Although this repair mechanism is the most efficient, it is error-prone, occasionally resulting in small insertions or deletions (indels), which can cause frameshifts and disrupt protein production. Thus, CRISPR can lead to the knockout of a gene (disrupting its protein expression), by creating a DNA double strand break that is erroneously repaired by the NHEJ mechanism.
Confused? Check out CRISPR Classroom’s CRISPR Foundations course for full details, images, and explanations on this process.
There are two important factors to consider when designing a guide RNA: specificity and target position.
When you design a CRISPR experiment, one of the most critical elements is the design of the gRNA. In order to create the most effective gRNAs, keep in mind the following tips.
Prior to designing your gRNA, determine the location of the genomic DNA you intend to target. Keep in mind gRNAs that do not match their targets exactly may have reduced efficiency. Make sure you check for any species- or cell-specific polymorphisms before you begin.
Next, locate all PAM motifs (NGG) in your area of interest. After discovering them, you can identify the ideal target location. Do not do this manually, you can use widely available online tools like CRISPOR.
Check that the gRNA matches the target DNA sequence, but equally as important, it should not match any other “off target” genomic sites. Tools like CRISPOR will automatically score your gRNA for off-target potential.
This gRNA design process is described in detail in CRISPR Classroom’s Gene Knockout: gRNA Design to Analysis mini course.
Experiment time! Test your CRISPR designs in cells.
After you have designed the gRNA, you co-deliver (co-transfect) the Cas9 plasmid or Cas9 protein (protein is recommended) and your gRNA into the chosen cell line. Lipid transfection, electroporation, or microinjection are all suitable transfection methods for Cas9/gRNA.
Screen for the genetic alterations you want after transfection. You may use Restriction Fragment Length Polymorphism assays (RFLP), Sanger sequencing, Next-Generation Sequencing, and/or Fluorescence-Activated Cell Sorting (FACS) to do so. The screening method that is best for your cell line will depend on the nature of your modifications and your experiment’s purpose.
The process of optimizing transfection to improve gene editing frequencies may require a bit of trial and error. Make sure you have chosen a dependable cell line to test your experiments. After your experiment is up and running in cell lines, you may move onto more difficult-to-edit primary cells, if needed.
Remember that immortalized cell lines are not only cheaper than primary cells, but also have recombination pathways that are often less stringent. Because of this, you should strive for a high gene editing efficiency in cell lines prior to working with primary lines because it can be much harder to achieve success.
Increase your Chances of Successful CRISPR Genome Editing
The efficiency of your CRISPR genome editing experiment is a mixture of planning and experience.
You can gain that experience by enrolling in CRISPR Classroom’s Gene Knockout: gRNA Design to Analysis minicourse to learn how to plan and analyze your own CRISPR experiments.
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