Genome editing has taken a leap forward as the benefits of clustered regularly interspaced short palindromic repeats (CRISPRs) technology become more widely adopted. Sean Ottewell reports.
Gene editing – the modification of an organism’s genome via a specific, targeted approach – is among the fastest growing applications for molecular biologists. Now, new tool kits are available to researchers that enable them to quickly make specific changes to DNA in a variety of cells.
For example, GeneArt CRISPR products from Thermo Fisher Scientific leverage the type II CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 system for genome editing.
This is an RNA guided DNA endonuclease system that has two key components, the Cas9 protein that serves as an DNA endonuclease and a non-coding RNA component called guide RNA (gRNA) that confers target specificity. The CRISPR/Cas9 system works in conjunction with endogenous repair mechanisms to alter DNA within a cell.
The CRISPR product portfolio is designed to offer many options for complete flexibility and ease of use (Fig. 1). These include, for example, GeneArt CRISPR nuclease mRNA, which is ready to transfect wild type Cas9 in mRNA format. The mRNA format is best for multiplexed screening of gRNA sequences, applications that require delivery by microinjection, and easier dosage optimisation of Cas9 protein to gRNA in the cell. Cas9 mRNA is co-transfected with one or more custom GeneArt CRISPR strings DNA encoding target-specific gRNA or in vitro transcribed gRNA which removes promoter constraints and is a better option for difficult to transfect cells such as induced pluripotent stem cells, or iPSCs (induced pluripotent stem cells).
A second option is the GeneArt CRISPR nuclease vector kits which offer reporter and enrichment tags for tracking gene editing experiments and are great for cloning and re-use of confirmed gRNA sequences. The GeneArt CRISPR nuclease vector with CD4 enrichment is a vector system for expression of the functional components of the CRISPR/Cas9 technology with a CD4 reporter. The CD4 reporter offers magnetic bead-based sorting/enrichment of Cas9 and CRISPR expressing cells using Dynabeads CD4 magnetic beads. Transfection efficiency can also be tracked using anti-CD4 fluorescent antibodies. The GeneArt CRISPR nuclease vector with orange fluorescent protein (OFP) reporter is a similar expression vector system with an (OFP) reporter. The OFP reporter allows for fluorescence-based tracking of transfection efficiency, as well as fluorescence activated cell sorting (FACS)-based sorting/enrichment of Cas9 and CRISPR expressing cells.
GeneArt CRISPRs can be used with other gene editing tools, such as the GeneArt Genomic Detection Cleavage Kit, providing a complete solution for design of synthetic genes, circuits and detection. Thermo Fisher can also design and clone CRISPRs for customers through the custom services team.
“CRISPRs provide a way to quickly and easily target those genomic sites a researcher wishes to modify in order to engineer cells,” said Helge Bastian, vice president for synthetic biology, life sciences solutions, Thermo Fisher Scientific. “Our new CRISPR kit format, along with our transfection reagents, offers a simplified workflow that enables the less-experienced user to easily carry out gene editing.”
Meanwhile Sigma Aldrich has launched Sigma CRISPRs, its latest offering to the global research community. “As the first company to commercially offer targeted genome editing technology nearly ten years ago, no one has more expertise in this field than Sigma Aldrich. This experience is especially important when it comes to crafting genome editing tools that possess the critical requirements of having specific targeting and robust cutting activity,” notes the company.
Pre-designed CRISPR sites that minimise off-targeting are available for coding regions of the human, mouse, and rat genomes. Custom designs for any other regions or species are always available upon request, says the company.
The company’s Sigma’s CRISPR paired nickases minimise off-target double-stranded breaks by requiring the independent binding of two separate gRNAs to a localised genomic region. In the presence of the Cas9-D10A nicking nuclease, the two gRNAs induce single-stranded breaks on opposite strands of the DNA, creating a functional double-stranded break.
When ordering Sigma′s CRISPR paired nickases for a specific target, researchers will receive two separate ready-to-use, transfection-grade plasmids expressing gRNA from the human U6 promoter.
OriGene’s latest contribution to the market is its new genome-wide gene-knockout kits using the CRISPR platform. The gene-centric research tool supplier’s new product line provides a complete solution for researchers to knock out a human gene at the chromosomal level. This, says the company, provides unprecedented convenience for genome editing using the CRISPR technology.
“CRISPR is a revolutionary tool for biomedical scientists. Our dream of a pair of simple accurate molecular scissor is now a reality. Launching genome-wide knock-out kits is only the very first step for OriGene to help harness the power of the CRISPR technology,” said Dr Wei-Wu He, ceo and chairman of OriGene.
“OriGene is in a perfect position to provide genome wide CRISPR tools to the research community. We provide a kit against every human gene locus, containing two gRNAs and a compatible donor vector with a functional cassette. In addition to gene knockout, this kit can also be used for promoter strength study for each gene locus,” added Dr Youmin Shu, senior vice president of molecular biology with the company.
Clontech licenses CRISPR/Cas9 technology from the Broad Institute
Clontech Laboratories, a wholly-owned subsidiary of Takara Bio, has entered into a non-exclusive licence agreement with the Broad Institute to access intellectual property related to the CRISPR/Cas9 gene editing system.
“Clontech is pleased to be able to provide innovative tools for CRISPR/Cas9 gene editing under our Guide-it brand,” commented general manager Carol Lou. “We believe that CRISPR/Cas9 technology represents a significant improvement over existing genome editing tools, reaching a new level of targeting, efficiency, and in particular ease of use. It is destined to become as valuable and widely utilised as RNAi; perhaps more so.”
Clontech’s initial product offering includes Guide-it kits for in vitro production and screening of single guide RNAs (sgRNAs) and a method to confirm the presence of targeted mutations using direct PCR and Clontech’s own Guide-it Resolvase enzyme.
The company develops, manufactures and distributes a wide range of life science research agents under both the Clontech and Takara brands. Key products include SMARTer cDNA synthesis kits for a variety of samples and applications, including NGS; high-performance qPCR and PCR reagents (including the TaKaRa Ex Taq, TaKaRa LA Taq, Titanium, and Advantage enzymes); RT enzymes and SMART library construction kits; the In-Fusion cloning system; tet-based inducible gene expression systems; Living Colours fluorescent proteins; and a range of Macherey-Nagel nucleic acid purification tools.
Launched in 2004, the Broad Institute is dedicated to discovering the molecular basis of major human diseases, developing effective new approaches to diagnostics and therapeutics, and disseminating discoveries, tools, methods, and data openly to the entire scientific community.
