CRISPR technology overview

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized the field of cell engineering.

With their highly flexible but specific targeting, CRISPR-Cas systems can be manipulated and redirected to become powerful tools for genome editing. CRISPR-Cas technology permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas systems is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell.

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The Power of CRISPR Genome Editing

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) system is the latest addition to the genome editing toolbox, offering a simple, rapid, and efficient solution. 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, and because the endonuclease cleavage specificity in CRISPR-Cas9 system is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell. The CRISPR-Cas9 system has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants.

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). 

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 1). 

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. 

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Figure 1. 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 GeneArt CRISPR-Cas9 genome-editing tools.

CRISPR-Cas9 system greatly simplifies genome editing and has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants. 

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Currently we are the only company to offer the complete suite of genome editing reagents.  We offer our state-of-the-art online Invitrogen™ CRISPR Search and Design Tool along with Invitrogen™ CRISPR-Cas9 editing products in four formats: CRISPR-Cas9 protein, CRISPR-Cas9 mRNA, CRISPR-Cas9 plasmid, and CRISPR library services. These gene-editing solutions are paired with optimal cell culture reagents, delivery methods, and analysis tools, based on your application and cell type.

Choose the optimized and validated toolset that’s right for you, and bypass the trial and error.

Delivery Method* Lipofectamine™ CRISPRMAX™ Lipofectamine™ MessengerMAX™ Lipofectamine™ 3000  
Format CRISPR Protein CRISPR mRNA CRISPR plasmid CRISPR Libraries
 
Product Name GeneArt™ Platinum™ Cas9 Nuclease GeneArt™ CRISPR Nuclease mRNA GeneArt™ CRISPR Nuclease Vector GeneArt™ CRISPR Arrayed Libraries
gRNA design Use the GeneArt CRISPR Search and Design Tool for optimal design and minimal off-target effects. Go to thermofisher.com/crisprdesign
gRNA synthesis GeneArt Precision gRNA Synthesis Kit GeneArt Precision gRNA Synthesis Kit DNA oligo cloned into plasmid n/a
Reporter-based enrichment Sold separately GeneArt™ Genomic Cleavage Selection Kit Sold separately GeneArt™ Genomic Cleavage Selection Kit ✓ included; all-in-one expression plasmid n/a
No promoter constraint CMV promoter
Ready-to-use Required cloning step Ready-to-use lentiviral particles
No random integration concern Concern Stable expression of CRISPR-Cas9 system
Controlled dosage  
Fast turnover    
Microinjection ready Larger payload size  
Multiplexing & screening capable Larger payload size High-throughput screening
Ready-to-act, stable RNP complex      
Modification options Knockout and knock-in Knockout and knock-in Knockout and knock-in Loss of function screening

*For the most efficient transfection of primary cells, stem cells, and difficult-to-transfect cells, use the Invitrogen  Neon  Transfection System.
Not sure which CRISPR-Cas9 format to use?  Let us help you.


Bench Tip Video: CRISPR/Cas Genome Editing Technology

In this Bench Tip Video, Dr. Mike Okimoto discusses CRISPR/Cas genome editing technology.CRISPR technology can be used to quickly and efficiently edit and manipulate a genomic locus in many different cell types and organisms. It can be used for a variety of genome modifications including gene deletion or knockout, knock-in, and knockdown.  Watch this video to learn how to set up a CRISPR experiment and the applications of CRISPR technology.

 

Need assistance with CRISPR gRNA design?

Our new simple, CRISPR Design tool provides access to >600,000 predesigned CRISPR gRNAs, options for de novo gRNA design, and recommendations based on potential off-target effects for each CRISPR sequence.

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