The transformative CRISPR-Cas9 technology is revolutionizing the field of genome editing. Able to achieve highly flexible and specific targeting, the CRISPR-Cas9 system can be modified and redirected to become a powerful tool for genome editing in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants. Thermo Fisher Scientific’s experts have created this collection of resources to give you the confidence to get started in gene editing or to continuously improve your research.

What is CRISPR technology?

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system is a simple, rapid, and efficient means to make changes to genetic sequences directly in cells. Derived from components of a simple bacterial immune system, the CRISPR-Cas9 system permits targeted gene cleavage and gene editing in a variety of cells.

Because the endonuclease cleavage specificity in the CRISPR-Cas9 system is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA (gRNA) sequence and delivering it along with the Cas9 endonuclease to a target cell.

How does CRISPR work?

The CRISPR-Cas9 system is composed of a short noncoding gRNA and a Cas9 nuclease (Figure 1). The gRNA has two molecular components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA). The gRNA unit guides the Cas9 protein to a specific genomic locus, where the Cas9 nuclease induces a double-stranded break at the specific genomic target sequence (Figure 2).

In bacteria, CRISPR loci are composed of a series of repeats separated by segments of exogenous DNA (of ~30 bp in length), called spacers. The repeat-spacer array is transcribed as a long precursor and processed within repeat sequences to generate small crRNAs that specify the target sequences (also known as protospacers) cleaved by the Cas9 nuclease. CRISPR spacers are then used to recognize and silence exogenous genetic elements at the DNA level.

Importantly, a specific three-nucleotide sequence immediately downstream of the 3’ end of the target region, known as the protospacer adjacent motif (PAM) sequence, must be present for cleavage to occur. This PAM sequence (NGG for Cas9) is present in the target DNA, but not the gRNA that targets it.

Following DNA cleavage using CRISPR-Cas9, the double-stranded break can be repaired by one of two aspects of the cell’s natural repair machinery (Figure 3).

  1. In the absence of a repair template, the nonhomologous end joining(NHEJ) process results in a heterogenous population of cells with different insertions or deletions (indels) around the gRNA-defined break. This process can be exploited to generate cell lines with random deletions around a specific sequence, producing a functional knockout.
  2. Alternatively, if a repair template is provided, a user-defined sequence change can be introduced at a specific locus within the genome via the error-free homology-directed repair (HDR) mechanism. This process can be used to overexpress a novel gene, create disease-relevant cell models, or tag endogenous genes with reportable moieties.

Tips and tricks for success when using CRISPR-Cas9 technology

When selecting CRISPR-Cas9 for your gene editing workflow, make sure you understand the factors affecting genome editing efficiency using this system. It is important to consider the design of your experiment, including the nuclease format and gRNAs used for cleavage, the transfection methods that will provide the most effective delivery of molecules into your cells, and the assays necessary to detect gene edits for appropriate validation of results.

Design and buildDeliverDetect and validate

Key considerations when designing your experiment:

  • Design and test three gRNAs for every target
  • Run bioinformatic analysis to choose gRNAs with minimal predicted off-target effects
  • Target early constitutive exons to disrupt the reading frame for knockout experiments
  • Ensure cleavage site is as close to the edit site as possible for knock-in experiments
  • Choose Cas9 nuclease best suited for the experiment

Key considerations when transfecting cells:

  • Start with optimal cell density and cell health
  • Understand the best method for transfecting your cells: lipid, electroporation, or viral
  • Optimize the ratio of their quantities when delivering multiple molecules
  • Transfect a validated control gRNA to test transfection efficiency and Cas9 activity
  • Deliver the repair template so that it is present when Cas9 is active  for knock-in experiments

Key considerations when detecting the edit:

  • Confirm gRNA cleavage efficiency quickly with a genomic cleavage detection (GCD) assay
  • Choose a detection method appropriate for the downstream application
  • Isolate clones if a genetically homogenous population is desired
  • Understand that the most efficient gRNA for indel-based knockout is typically the most efficient gRNA for knock-in
  • Verify lack of off-target editing using targeted sequencing
Screen capture of infographic which outlines various tips and tricks for using CRISPR-Cas9

Tips and tricks for CRISPR success

This infographic will serve as your guide to CRISPR-Cas9 with best practices, pro tips, and troubleshooting advice.


Available CRISPR technology

Thermo Fisher Scientific offers a complete suite of genome editing reagents (Figure 4). These gene editing solutions are paired with optimal cell culture reagents, delivery methods, and analysis tools, based on your application and cell type.

Diagrams illustrating Cas9 nuclease and gRNA formats
Figure 4. Available CRISPR-Cas9 nuclease and gRNA formats.

Featured CRISPR tools and resources

For Research Use Only. Not for use in diagnostic procedures.