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.
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.
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).
Figure 3. CRISPR-Cas9 double-stranded break and repair pathways. Cleavage occurs on both strands, three base pairs upstream of the NGG in the PAM sequence on the 3’ end of the target sequence.
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 build||Deliver||Detect and validate|
Key considerations when designing your experiment:
Key considerations when transfecting cells:
Key considerations when detecting the edit:
This infographic will serve as your guide to CRISPR-Cas9 with best practices, pro tips, and troubleshooting advice.
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.
Intended as an introduction to genome editing, the resource guide covers available technologies along with methods for design, delivery, and detection of edited cells.
Step-by-step CRISPR protocols have been optimized for maximum efficiency, viability, and reproducibility across a broad range of cell types and gene targets.
The Invitrogen TrueDesign Genome Editor is a free online tool that enables you to easily design, select, and order reagents for accurate and precise gene editing experiments.
Invitrogen TrueGuide Synthetic gRNAs are ready-to-transfect synthetic gRNAs designed and validated to provide consistent high efficiency editing.
Access a targeted collection of scientific application notes, publications, videos, webinars, and scientific posters for genome editing.
A complete guide for gene editing in PSCs, including all the tools and protocols needed to design, deliver, and screen for preferred gene edits in PSCs.
For Research Use Only. Not for use in diagnostic procedures.