Pooled CRISPR/shRNA Library Construction
VectorBuilder can help you build custom pooled CRISPR or shRNA libraries to perform large-scale functional screens. We have extensive experience in constructing high-quality pooled libraries utilizing our optimized vectors. We can deliver your library as E. coli stock, DNA, or packaged virus, depending on your needs. Our custom libraries are fully validated by next generation sequencing so you know exactly what you get.
How to obtain pooled CRISPR/shRNA library construction service?
Submit your needs to us by clicking the Send Design Request button on the homepage. Our scientists will send you a link to a service proposal including price, turnaround and other relevant information. If you are satisfied with the proposal, you can put it in your shopping cart and follow instructions there to place an order.
Pooled CRISPR/shRNA libraries and their use in genetic screen
Pooled CRISPR libraries
Pooled CRISPR libraries are a powerful tool for performing large-scale or genome-wide functional screens of coding or noncoding regions involved in particular pathways, diseases, cell responses to drug treatment, developmental processes, gene regulation, etc. The table below lists and compares commonly used CRISPR-based screens:
|Screen Type||Mechanism||Target Location||Application|
|Knockout||Wildtype Cas9 or Cas9 nickase is used to cut DNA. When cells attempt to repair DNA breakage, random mutations can be introduced at target sites.||Primarily coding regions||Mainly for functional screens of coding genes|
|CRISPRa (activation)||Catalytically inactive Cas9 (dCas9) fused to a transcriptional activator (e.g. VP64) is used to activate genes regulated by target sites.||Typically promoter regions and other noncoding regulatory regions||Gain-of-function screen of coding genes, or screens for regulatory functions of noncoding regions|
|CRISPRi (inhibition)||Catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor (e.g. KRAB) is used to inhibit genes regulated by target sites.||Typically promoter regions and other noncoding regulatory regions||Loss-of-function screen of coding genes, or screens for regulatory function of noncoding regions|
For typical knockout screens, the pooled CRISPR library can be constructed in the format of either 1-vector or 2-vector systems. In 1-vector systems, Cas9 or Cas9 variant is co-expressed with gRNA from the same vector, while in 2-vector systems, Cas9 or Cas9 variant is expressed from one vector while gRNA is expressed on the other vector. Alternatively, in 2-vector systems, gRNA-expressing vectors can be introduced into cell lines that stably express Cas9. The advantages of using 1-vector systems is that it avoids co-transfection/transduction of two different vectors into cells and it is not limited to the availability of Cas9-expressing stable cell line. However, it is less versatile and can be less efficient in cloning and virus packaging than 2-vector systems.
Pooled shRNA libraries
Pooled shRNA libraries can serve as a powerful and cost-efficient tool for performing large-scale or genome-wide loss-of-function screens in mammalian cells. A major drawback of CRISPR-based knockout screens is the high cell-to-cell variability in knockout effect for a given target site due to the stochastic nature of CRISPR-introduced mutations. In contrast, pooled shRNA-based screens generally yield uniform knockdown effect from cell to cell for a given target gene.
In addition to custom pooled shRNA library construction, VectorBuilder offers high-quality premade pooled shRNA libraries targeting human and mouse genes. For each species, we provide ready-to-use lentivirus libraries at two scales: Whole Genome (~19,000 RefSeq genes) and Elite Gene (~2,000 most frequently cited genes on PubMed Central). Where possible, each gene is targeted by 5-6 different shRNAs. These libraries have been fully validated by next-generation sequencing and functional assays.
Overview of services
|Available Services||Brief Description||Price||Turnaround|
|Design custom library construction strategy||Free||1-4 days*|
|Custom pooled CRISPR or shRNA library construction||Includes array-based oligo synthesis of gRNA/shRNA, cloning of gRNA/shRNA into desired vector backbone, and plasmid pool validation by next-generation sequencing (NGS). **||From $5,000||6-8 weeks|
|Amplification of premade plasmid library pool***||If you have a premade library in the form of a plasmid pool (e.g. CRISPR plasmid pool obtained from Addgene), we can package it into virus for you. An amplification step is often needed to obtain enough DNA for virus packaging. For this, the plasmid pool is transformed into E. coli cells with an average of >100x representation, and plasmid DNA is prepared for virus packaging.||From $200||3-5 days|
|Virus packaging of pooled library||Please click here to view details of virus packaging services.|
|NGS validation of premade plasmid library pool||We can validate the quality of your premade plasmid library pool by NGS before and/or after amplification. This includes NGS library preparation from plasmid pool, Illumina sequencing (>500x coverage), and data analysis.||From $500 per plasmid pool||2-4 weeks|
|NGS deconvolution of post-screening sample||Includes NGS library preparation from genomic DNA of screened cells, Illumina sequencing (> 500x coverage), and data analysis.||From $800 per sample||2-5 weeks|
|Other custom library construction||Please inquire|
* If design of gRNA or shRNA is needed, turnaround time may be longer for specific projects.
** The default deliverable is E. coli glycerol stock.
*** For user-supplied premade plasmid library pool, we cannot make any guarantees regarding the complexity or uniformity of the library.
Detailed description of services
Design custom library construction strategy
If you don’t have the actual sequences of gRNA or shRNA, we can help you design gRNA or shRNA pool for your list of target genes or genomic regions using our optimized pipeline and powerful computational resources. Depending on your research needs, you can choose from our various validated vector backbones for constructing the library, such as lentivirus, adeno-associated virus (AAV), piggyBac and regular plasmid. For 2-vector CRISPR libraries, we can also design and construct Cas9 or Cas9 variant expression vectors that are suitable for your screening strategy.
Custom pooled CRISPR/shRNA library construction
We use a chip-based approach to synthesize high-quality gRNA or shRNA oligo pools with a low error rate. The oligos are then PCR amplified with a minimal number of cycles and efficiently cloned into the desired vector backbone with an average of >100x representation. E. coli cells harboring CRISPR/shRNA library plasmids are propagated and harvested in exponential growth phase to make glycerol stocks. To assess the quality of the library, NGS is performed on the gRNA/shRNA plasmid pool, with sequencing depth at >500x coverage. NGS data are analyzed and all designed gRNA/shRNA with normalized abundance in the pool is reported.
Amplification of premade plasmid pool
We can amplify premade plasmid library pool from other sources (e.g. Addgene) for you. We will first transform pooled plasmids into super-competent cells with an average of >100x representation. E. coli cells are harvested in exponential growth phase to make glycerol stocks. If downstream virus packaging is requested, plasmid DNA will be isolated by maxi prep. We count the number of colonies after transformation to estimate the actual average gRNA/shRNA fold representation.
We can perform NGS validation of your plasmid pool before and/or after transformation. Please note that for plasmid pool not constructed by VectorBuilder, we guarantee >100x average representation as estimated by counting colonies, but we cannot guarantee the complexity or uniformity of the library because we have no control over the composition of your premade plasmid pool.
Virus packaging of pooled library
For CRISPR/shRNA screens, you may need to deliver the pooled library into cells through virus, such as lentivirus or adeno-associated virus. VectorBuilder has developed a range of proprietary technologies and reagents that 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 cloning services. As a result, we have a growing base of highly satisfied customers who come back to us time after time for their cloning and virus packaging needs.
View more information of virus packaging services >>
NGS deconvolution of post-screening sample
Not sure how to identify hits by NGS after conducting your screen? You can simply send the genomic DNA of your samples to VectorBuilder, and we will prepare the NGS libraries, perform high-throughput sequencing, analyze the data, and deliver the accurate counts of gRNA/shRNA in each sample to you in just a few weeks.
We will notify you by email when the sequencing is completed, and provide you with an FTP link for downloading raw sequencing data and analyzed data. We will keep your data on our FTP site for 3 months for free, but you can request to store your data for longer.
Other custom library construction
If you need custom-built vector libraries to perform large-scale screens other than CRISPR/shRNA, be it phage display, two-hybrid, or gene expression libraries, VectorBuilder can help. Just submit your needs through Send Design Request, and our scientists will get back to you within 1-2 days with detailed service proposal.