CRISPR-Cas9 is revolutionizing the field of genome editing
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. We've put together a collection of resources that we hope will give you the confidence to get started and to continuously improve your research.
The system that comprises clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) is the latest addition to the genome editing toolbox, offering a simple, rapid, and efficient tool. Derived from components of a simple bacterial immune system, the CRISPR-Cas9 system permits targeted gene cleavage and gene editing in a variety of eukaryotic 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 your target cell.
The CRISPR-Cas9 system is composed of a short noncoding guide RNA (gRNA) that 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 via base pairing between the crRNA sequence and the target sequence (Figure 1).
Figure 1. The CRISPR gRNA and Cas9 Protein.
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 Cas9 protein, the nuclease component of CRISPR system. CRISPR spacers are then used to recognize and silence exogenous genetic elements at the DNA level. Essential for cleavage is a three-nucleotide sequence motif (NGG) immediately downstream on the 3’ end of the target region, known as the protospacer-adjacent motif (PAM). The PAM is present in the target DNA, but not the crRNA that targets it (Figure 2).
Figure 2. The CRISPR-Cas9 System.
Upon binding to the target sequence, the Cas9 protein induces a specific double-strand break. Following DNA cleavage, the break is repaired by cellular repair machinery through non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. With target specificity defined by a very short RNA-coding region, the CRISPR-Cas9 system greatly simplifies genome editing (Figure 3).
Figure 3. A CRISPR-Cas9 targeted double-strand break. Cleavage occurs on both strands, 3 bp upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3’ end of the target sequence.
Available CRISPR-Cas9 genome-editing tools
Thermo Fisher Scientific offers a complete suite of genome editing reagents. 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 the technologies available today for gene editing, along with methods for design, delivery and detection of edited cells.
CRISPR Validated Protocols
Step-by-step CRISPR protocols have been optimized for maximum efficiency, viability, and reproducibility across a broad range of cell types and gene targets.
TrueDesign Genome Editor software
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.
TrueGuide Synthetic gRNA Order Tool
Invitrogen TrueGuide Synthetic gRNAs are ready-to-transfect synthetic gRNAs designed and validated to provide consistent high efficiency editing.
Journal of Biotechnology publication: Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA
Demonstrated protocol: CRISPR-Cas9 microinjection in mice and zebrafish embryos
Strategies and tools for optimal editing efficiencies in mice and zebrafish. This step-by-step protocol was designed to guide you through efficient CRISPR embryo microinjections in mice and zebrafish.
Read the what, why, and how of CRISPR genome editing and its use in myriad endeavors including cell and protein analysis.
CRISPR and TALEN tools and technologies are changing the way we do gene editing today and in the future, but what are the differences between these two? And when would you use one technology over the other? This article addresses these questions and more.