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.

Click the links below to view the detailed description of our CRISPR offerings:
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.

Click to read more information on the mechanism of CRISPR genome editing

CRISPR delivery approach

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

  • Cas9 and gRNA expression plasmid
  • Cas9 and gRNA expression virus (i.e. lentivirus, AAV, adenovirus, etc.)
  • Mixture of Cas9 mRNA and gRNA
  • Preformed ribonucleoprotein (RNP) between Cas9 protein and gRNA
Figure 2. Four methods for delivering CRISPR components into cells.

The table below lists key advantages and disadvantages for each delivery approach which can be used as a reference to help you decide the most suitable approach for your experiments:

Delivery Approach Advantages Disadvantages
Cas9 and gRNA plasmid
  • Delivered by chemical transfection or electroporation which is simple and low-cost.
  • Able to achieve high-level expression of Cas9 protein and gRNA for a few days.
  • Cas9 and gRNA can be delivered in all-in-one plasmid, therefore circumventing the need for transfecting multiple components.
  • Plasmids as a reagent can be generated and regenerated very cheaply and in very large quantities, making them suitable for CRISPR experiments that require a lot of the reagent.
  • Efficiency of plasmid transfection varies widely between cell types.
  • Dependent on cell-type specific promoter activity.
  • Largely limited to in vitro applications.
  • Risk of random integration of plasmid DNA into the host genome.
  • Risk of off-target effects due to high-level Cas9 and gRNA expression.
Cas9 and gRNA virus
  • Suitable for genome editing applications in difficult-to-transfect cells.  
  • Cas9 and gRNA can be delivered in a single all-in-one viral vector, therefore circumventing the need for transduction of multiple components.
  • Can achieve prolonged or stable expression of Cas9 protein and gRNA.
  • Requires virus packaging which is technically challenging and time-consuming, unless outsourced to VectorBuilder.
  • Dependent on cell-type specific promoter activity.
  • Elevated risk of insertional mutagenesis.
  • Risk of off-target effects due to prolonged Cas9 expression.
Mixture of Cas9 mRNA and gRNA
  • Delivered by chemical transfection or electroporation which is simple.
  • Rapid genome editing as no transcription is needed for Cas9 and gRNA expression.
  • Independent of cell-type specific promoter activity.
  • No risk of random insertion into host genome.
  • Editing activity occurs only transiently, after which it drops off quickly as the transcripts get degraded inside the host cells.  
Preformed Cas9-gRNA RNP complex
  • Can be delivered by electroporation, making it suitable for difficult-to-transfect cells.
  • Rapid editing as neither transcription nor translation is needed for Cas9 and gRNA expression.
  • Independent of cell-type specific promoter activity.
  • No risk of random insertion into host genome.
  • Electroporation-based RNP delivery requires expensive hardware and consumables.
  • Editing activity occurs as a rapid pulse, after which it drops off quickly as gRNA-Cas9 RNP is degraded inside the host cells.
  • Limited by the availability of purified Cas9 protein.
Our CRISPR offerings
Custom CRISPR vectors

CRISPR-mediated genome editing requires the target cells to co-express Cas9 and target site-specific gRNA at the same time. Using our highly intuitive online vector design platform, you can choose from over 30 vector backbones (non-viral, viral or transposon) and unlimited combinations of vector components (promoters, Cas9 variants, fluorescent and drug-selection markers) to express Cas9 and gRNA in different manners. Our gRNA database allows you to easily pick appropriate guide sequence for your target genes without looking for and analyzing the target sites on your own. We offer all-in-one as well as dual vector systems for Cas9 and gRNA expression in regular plasmid, lentivirus, AAV, adenovirus and PiggyBac.

Our comprehensive collection of CRISPR vectors also includes gene targeting donor vectors for serving as template DNA to guide precise sequence changes such as point mutations and large fragment knockins at target sites through HDR. Additionally, we offer CRISPRa and CRISPRi vectors for achieving transcriptional activation or inhibition of target genes within their endogenous loci.

Plasmid DNA preparation, RNA preparation and virus packaging can be purchased as downstream services when adding CRISPR vectors into shopping cart.

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:

Vector System Vector Name Vector ID 価格 (税抜き)
hCas9 expression vector (regular plasmid) pRP[Exp]-
mCherry/Hygro-CBh>hCas9
VB170315-1076pcv 23000円
hCas9 expression vector (lentivirus) pLV[Exp]-
CBh>hCas9:T2A:Hygro
VB160923-1033trt 23000円
SaCas9 expression vector (AAV) pAAV[Exp]-
CMV>SaCas9
VB180125-1089ffa 23000円
hCas9 expression vector (adenovirus) pAV[Exp]-CBh>hCas9 VB180820-1032mbq 23000円
hCas9 expression vector (piggyBac) pPB[Exp]-mCherry/Hygro-CBh>hCas9 VB181219-1015jaf 23000円
Scramble gRNA control vector (regular plasmid) pRP[gRNA]-EGFP/Puro-U6>Scramble_gRNA VB171211-1212bte 15500円
Scramble gRNA control vector (lentivirus) pLV[gRNA]-EGFP/Puro-U6>Scramble_gRNA VB160602-1127urj 15500円
Scramble SagRNA control vector (AAV) pAAV[gRNA]-EGFP/Puro-U6>Scramble_SagRNA1 VB181225-1325hfs 15500円
Scramble gRNA control vector (adenovirus) pAV[gRNA]-EGFP-U6>Scramble_gRNA1 VB180416-1105sbw 15500円
Scramble gRNA control vector (piggyBac) pPB[gRNA]-EGFP/Puro-U6>Scramble_gRNA1 VB181225-1329xyy 15500円
hCas9 and scramble gRNA coexpression vector (regular plasmid) pRP[CRISPR]-EGFP/Puro-hCas9-U6>Scramble_gRNA1 VB180725-1096pqd 15500円
hCas9 and scramble gRNA coexpression vector (lentivirus) pLV[CRISPR]-hCas9/Puro-U6>Scramble_gRNA1 VB180522-1197bhe 15500円
SaCas9 and scramble SagRNA coexpression vector (AAV) pAAV[CRISPR]-SaCas9-U6>Scramble_SagRNA VB170425-1003ytn 15500円
hCas9 and scramble gRNA coexpression vector (adenovirus) pAV[CRISPR]-hCas9/EGFP-U6>Scramble_gRNA1 VB181225-1306tra 15500円
dCas9-SAM activator MS2/P65/HSF1 expression vector (lentivirus) pLV[Exp]-EF1A>MS2/P65/HSF1/Hygro VB170424-1011udy 23000円
dCas9-SAM activator dCas9/VP64 expression vector (lentivirus) pLV[Exp]-EF1A>dCas9/VP64/Bsd VB170424-1009jup 23000円
dCas9-SAM scramble msgRNA control vector (lentivirus) pLV[msgRNA]-EGFP/Puro-U6>Scramble_gRNA VB180124-1069tzr 15500円
dCas9-KRAB-MeCP2 expression vector (lentivirus) pLV[Exp]-CBh>dCas9/
KRAB/MeCP2:T2A:Hygro
VB190101-1021vwr 23000円
CRISPRウイルス 

Lentivirus, AAV and adenovirus are widely used to deliver CRISPR components into mammalian cells. VectorBuilder offers premium-quality virus packaging services for lentivirus, AAV and adenovirus for achieving highly efficient CRISPR targeting in difficult-to-transfect cells. Our proprietary technologies and reagents have greatly improved virus packaging protocols in terms of titer, purity, viability and consistency. Our packaging protocols are also optimized for the viral vector systems used in our vector construction services. As a result, we have a growing base of highly satisfied customers who come back to us again and again for their cloning and virus packaging needs.

レンチウイルスパッケージングサービス仕様:

サイズ 推奨する使用系 タイターと仕様 価格 (税抜き) 作業日数
パイロット 培養細胞 >108 TU/ml, 10x25 ul, HBSS buffer 66,000円 8-16 日
中容量 培養細胞 >108 TU/ml, 10x100 ul, HBSS buffer 92,000円 8-16 日
大容量 培養細胞 >109 TU/ml, 10x100 ul, HBSS buffer 144,000円 8-16 日
超純粋中容量 培養細胞とin vivo >109 TU/ml, 10x50 ul, HBSS buffer 170,000円 8-16 日
超純粋大容量 培養細胞とin vivo >109 TU/ml, 10x100 ul, HBSS buffer 209,000円 8-16 日

AAV パッケージングサービス仕様:

サイズ 推奨する使用系 タイター*と仕様  価格 (税抜き) 作業日数
パイロット 培養細胞 >1011 GC/ml, 10x25 ul, PBS buffer 66,000円 10-20 日
中容量 培養細胞 >1011 GC/ml, 10x100 ul, PBS buffer 85,500円 10-20 日
大容量 培養細胞 >1012 GC/ml, 10x100 ul, PBS buffer 144,000円 10-20 日
超純粋パイロット 培養細胞と in vivo >1013 GC/ml, 4x25 ul, PBS buffer 163,500円 20-30 日
超純粋中容量 培養細胞と in vivo >1013 GC/ml, 10x50 ul, PBS buffer 229,000円 20-30 日
超純粋大容量 培養細胞と in vivo >1013 GC/ml, 10x100 ul, PBS buffer 345,500円 20-30 日

アデノウイルスパッケージングサービスの仕様:

サイズ 推奨する使用系 タイター*と仕様 価格 (税抜き) 作業日数
パイロット Cell culture >1010 PFU/ml, 10x25 ul, HBSS buffer 66,000円 20-32 日
中容量 Cell culture >1010 PFU/ml, 10x100 ul, HBSS buffer 92,000円 20-32 日
大容量 Cell culture >1011 PFU/ml, 10x100 ul, HBSS buffer 144,000円 20-32日
超純粋大容量 Cell culture and in vivo >1012 VP/ml, 10x100 ul, GTS buffer 209,000円 22-36 日

Note:

a. Estimated turnaround is the time from production initiation to completion. It does not include waiting time for any customer-supplied materials (e.g. template DNA), QC of such materials, and transit time for shipping final deliverables to the customer.

b. Our titer guarantee only applies to vectors for which the region being packaged into virus does not exceed the cargo capacity of the virus. For lentivirus, it is 9.2 kb (from Δ5’ LTR to ΔU3/3’ LTR). For AAV, it is 4.7 kb (from 5' ITR to 3' ITR). For adenovirus, it is 38.7 kb (from 5' ITR to 3' ITR).

c. We are not able to guarantee titer for the following vectors:

  • vectors containing sequences that could adversely affect the packaging process such as toxic genes (e.g. proapoptotic genes), genes that compromise the integrity of packaging cells or virus (e.g. membrane proteins that cause cell aggregation), and sequences prone to rearrangements or secondary structures (e.g. repetitive or highly GC-rich sequences);
  • customer-supplied vectors as we have no control over their quality.

Click here to view detailed information on our virus 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:

mRNA 濃度と容量 価格 (税抜き) 作業日数
hCas9 mRNA >500 ng/ul, 25 ul, nuclease-free water, sterile 47000円 3-5 日
Cas9(D10A) mRNA >500 ng/ul, 25 ul, nuclease-free water, sterile 47000円 3-5 日
Custom gRNA* >500 ng/ul, 25 ul, nuclease-free water, sterile 47000円 3-5 日

* Construction of gRNA in vitro transcription vector has an additional $99 charge and 7-14 days turnaround.

Cas9 タンパク質

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:

精製タンパク質 濃度と容量 価格 (税抜き) 作業日数
SpCas9 protein 100 ug 89,000円 5-7 日
SpCas9(D10A) nickase protein 100 ug 89,000円 5-7 日
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:

Reagent 価格 (税抜き) Turnaround
ssODN (normally 120-200 bp) 47,000円 2-3 weeks
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.

Click here for detailed information on our library construction services

In addition to the custom pooled CRISPR libraries, we also offer pre-made dual-gRNA lentivirus libraries for whole-genome knockout screens in human and mouse. Dual-gRNA libraries are far more powerful than single-gRNA libraries for knockout screens because the introduction of large deletions by these libraries can have much higher efficiencies in generating loss-of-function mutations. The human and mouse dual-gRNA libraries are made in the form of ready-to-use high-titer pooled lentivirus targeting 20,048 human genes and 20,493 mouse genes, respectively. Wherever possible, each gene is targeted redundantly by 4-6 different gRNA pairs in separate vectors.

Highlights of our pre-made dual-gRNA CRISPR libraries:

  • 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:

Product name No. of genes No. of gRNA pairs Scale* Catalog No. 価格 (税抜き)
Human Whole-Genome Dual-gRNA Lentivirus Library 20,048 91,926

Medium

(>1.0x108 TU/ml, 1 ml)

LVM(Lib190505-1046fgb) 534,000 円1,003,000 円

Plus

(>1.0x108 TU/ml, 5 ml)

LV5M(Lib190505-1046fgb) 1,119,000 円2,128,000 円
Mouse Whole-Genome Dual-gRNA Lentivirus Library 20,493 90,344

Medium

(>1.0x108 TU/ml, 1 ml)

LVM(Lib190505-1050kpm) 534,000 円1,003,000 円

Plus

(>1.0x108 TU/ml, 5 ml)

LV5M(Lib190505-1050kpm) 1,119,000 円2,128,000 円

Click here for detailed information on our pre-made dual-gRNA CRISPR 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!

  • Gene knockout through gene disruption
    • Random frameshift resulting from NHEJ DNA repair at single cut site
    • Exon deletion between two cut sites
  • Introduction of point mutation
  • Fragment knockin
    • Small tag insertion (< 60 bp)
    • Large fragment insertion (up to 4-5 kb)
  • CRISPR gene activation
  • CRISPR gene inhibition
  • CRISPR libraries (KO, CRISPRa, CRISPRi, barcoding, etc.)
  • Stable cell line generation
    • Cas9-expressing cell line
    • iPSC genome editing
    • Cancer cell genome editing

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

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