CRISPRシステムを使った遺伝子編集

VectorBuilder offers a variety of CRISPR products and services for in vitro and in vivo genome editing experiments with unbeatable prices and rapid turnaround. Our CRISPR offerings range from off-the-shelf reagents that are ready for transfection or transduction to custom-made CRISPR vectors, available in multiple delivery formats (i.e. plasmid, virus, RNA). We can do virus packaging of all major viral types (i.e. lentivirus, AAV, adenovirus) to deliver your CRISPR components efficiently into difficult-to-transfect cells. Additionally, we are specialized in building high-quality CRISPR libraries for knockout, gene activation, gene inhibition and other CRISPR screens. Our uniquely designed and well validated whole genome dual-gRNA knockout libraries are powerful tools for gene functional screens in human and mouse. With our extensive experience in molecular cloning, gene delivery and gene targeting, VectorBuilder provides one-stop solutions to all your CRISPR needs.

Highlights of VectorBuilder's CRISPR products & services

• Highly intuitive online vector design platform with whole-genome gRNA database implemented for easy and quick CRISPR design
• Rich collection of vector backbones (i.e. regular plasmid, lentivirus, AAV, adenovirus, piggyBac) and vector components (i.e. promoters, Cas9 variants, fluorescent and drug-selection markers)
• CRISPR components available in a variety of delivery formats (i.e. plasmid, virus, Cas9 mRNA + gRNA, Cas9-gRNA RNP complex)
• Versatile CRISPR library construction options
• Robust quality, fast turnaround and competitive pricing
• Powerful technical support for experimental design, data analysis and troubleshooting

CRISPR-mediated genome editing

The conventional use of CRISPR system contains two components: Cas9 protein and guide RNA (gRNA). The most commonly used Cas9 is engineered from Streptococcus pyogenes (a.k.a. SpCas9 or hCas9). It is an RNA-guided DNA nuclease which can generate double-stranded breaks (DSBs) at target sites (Figure 1). Another widely used Cas9 variant, Cas9 “nickase” (e.g. Cas9(D10A)), generates only single-stranded cuts in DNA. If Cas9 nickase is used in conjunction with two gRNAs targeting the two opposite strands flanking a single target region, then the nickase will generate two single-stranded cuts, one on each strand, resulting in DSBs at the target region. Once DSBs are generated by the CRISPR system, cells will undergo nonhomologous end-joining (NHEJ) pathway to repair the DNA breaks, which usually results in small random deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), they may lead to functional gene knockout. Alternatively, and less efficiently, DSBs can be repaired via homology-directed repair (HDR) pathway, with the presence of exogenous donor DNA template, which is co-introduced with the CRISPR components. This can result in replacement of the target genomic DNA sequence with donor DNA sequence, generating precise sequence changes at the target site, such as point mutations or knockin of the donor DNA sequence. The donor DNA template can be single-stranded oligo DNA nucleotide (ssODN) or dsDNA fragment (usually linearized plasmid DNA). ssODNs are suitable for introducing point mutations or small tag insertions, while dsDNA fragments are widely used to introduce large fragment knockins.

Figure 1. Mechanism of CRISPR-induced DNA repair.

CRISPR delivery approach

CRISPR components can be introduced into mammalian cells via different approaches (Figure 2), including:

Our collection of CRISPR vectors include:

Note: AAV has a cargo capacity of 4.7 kb. The commonly used SpCas9 derived from Streptococcus pyogenes is 4.2 kb, which can hardly fit into AAV when combined with other components such as promoter, polyA signal sequence, and the gRNA expression cassette. Our standard AAV CRISPR system therefore utilizes the shorter SaCas9 (3.2 kb) derived from Staphylococcus aureus. Please note that the PAM sequence recognized by SaCas9 is NNGRR or NNGRRT (preferred), whereas the PAM for SpCas9 is NGG. We can also construct AAV vectors compatible with SpCas9 upon request.

Pre-made CRISPR vectors

In addition to custom CRISPR vectors, VectorBuilder also offers a panel of pre-made Cas9 expression vectors, control gRNA vectors and helper vectors for CRISPRa and CRISPRi. Our off-the-shelf vectors are available for immediate shipment as E. coli stocks. Plasmid DNA preparation, RNA preparation and virus packaging can be purchased as downstream services when adding pre-made vectors into shopping cart.

Our collection of pre-made CRISPR vectors include:

Price, turnaround and scales of our AAV packaging services：

RNA preparation for Cas9 mRNA and gRNA

VectorBuilder can provide transfection-ready and microinjection-ready Cas9 mRNA and gRNA specifically designed against user-selected target sites for easy RNA-based delivery of CRISPR components into mammalian cells. Cas9 mRNA is available for both the wildtype nuclease (hCas9) and the Cas9 nickase (Cas9(D10A)). We follow the algorithm developed in Feng Zhang’s lab to calculate specificity score and apply a set of empirical rules in CRISPR targeting to design the optimal gRNA for targeting user-selected genes/sites.

Price and turnaround of our CRISPR RNA products:

Cas9 protein

VectorBuilder offers purified wildtype Streptococcus pyogenes Cas9 protein (SpCas9) and the Cas9 nickase (Cas9(D10A)) for preparing preformed Cas9-gRNA RNP to deliver CRISPR components into mammalian cells. The RNP-mediated approach can be used to deliver desired gRNA in conjunction with Cas9 protein. Additionally, preformed RNPs can also be delivered together with donor templates (ssODN or dsDNA) to induce HDR-mediated DNA repair at CRISPR target site for precise genome editing.

Price and turnaround of our Cas9 protein products:

Donor DNA for precise genome editing

VectorBuilder offers donor DNA templates in the form of ssODN or dsDNA from linearized plasmid for guiding HDR-based DNA repair at CRISPR cleavage sites to introduce precise DNA sequence changes at target sites. The introduced changes include point mutations and small or large fragment knockins. While ssODNs are utilized for inserting short DNA sequences (< 60 bp) such as small tags or restriction enzyme sites at target sites, dsDNA donors are utilized for targeted knockin of larger sequences (up to 4-5 kb) such as fluorescent tags and other reporters.

Price and turnaround of our donor DNA products:

Pooled CRISPR libraries

Pooled CRISPR libraries are powerful tools for performing large-scale functional screens of coding or noncoding regions involved in particular pathways, diseases, cell responses to drug treatment, developmental processes, gene regulation, etc. VectorBuilder specializes in the custom design and construction of a variety of pooled libraries for commonly used CRISPR knockout libraries for functional screens of coding genes, CRISPRa libraries for gain-of-function screens of coding genes or regulatory function screens of noncoding regions and CRISPRi libraries for loss-of-function screens of coding genes or for screening regulatory function of noncoding regions. Additionally, we can also build CRISPR barcode libraries for single cell-based screens and other pooled libraries utilizing CRISPR technology.

We can deliver your library as E. coli stock, plasmid DNA pool, or packaged virus, depending on your needs. Our custom libraries are fully validated by next-generation sequencing so that you know exactly what you get.

• Unique dual-gRNA lentiviral vector design
• Whole-genome and high-coverage targeting
• Validation of library quality by NGS
• High uniformity
• Available as ready-to-use high-titer lentivirus
• Dual EGFP/Puro marker for efficient and versatile selection or tracking of positively transduced cells

Price and turnaround of our pre-made dual-gRNA libraries:

CRISPR solutions

The versatility of the CRISPR system makes it suitable for a wide variety of genome editing applications in mammalian cells. Our technical team with extensive experience in molecular biology can provide complete solutions for all your CRISPR genome editing projects from experimental design to generation of the ready-for-use reagents. We can help you with the common CRISPR applications listed below, and we can work with you on any novel CRISPR applications. Send us a design request immediately to get a free service proposal!

Click here for a detailed information on our stable cell line generation services

VectorBuilder's online “Learning Center” contains rich educational resources to facilitate you to successfully plan, execute and troubleshoot your CRISPR experiments.

Click here to read guides on CRISPR vector systems

Click here to read guides on CRISPR components

Click here to read CRISPR-related FAQs