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 do I obtain pooled CRISPR/shRNA library construction service?

Submit your needs to us by clicking the Send Design Request button on our 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 add it to your shopping cart and follow the instructions there to place your 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, a 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 gRNAs are expressed on separate vectors. Alternatively, in 2-vector systems, gRNA-expressing vectors can be introduced into cell lines that stably express Cas9. The advantages of using the 1-vector systems are that it avoids co-transfection/transduction of two different vectors into cells and it is not limited to the availability of a 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 the 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 across cells 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.

View more information of premade pooled shRNA libraries

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 preliminary validation of the plasmid pool by Sanger sequencing. ** From $3,000 4-6 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 E. coli glycerol stocks are prepared for long-term storage. ** From $300 3-5 days
Library DNA preparation >1 ug/ul, 150 ul, 1x TE buffer, endotoxin-free, sterile $109 4-6 days
Virus packaging of pooled library Please click here to view detailed scales 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 $300 3-4 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 $250 per sample 5-7 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.
*** If the premade plasmid pool is supplied by the customer, please send us the vectors following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur a $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC. For customer-supplied premade plasmid library pools, we cannot provide any guarantees regarding the complexity or uniformity of the library.
**** The charge for packaging library plasmid is 1.5-fold of the charge for packaging plasmid from a single clone.

Detailed description of services
Design custom library construction strategy

If you need assistance in designing your gRNA or shRNA sequences for your list of target genes or genomic regions, we can help you design them 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 our lentivirus, adeno-associated virus (AAV), piggyBac and regular plasmids. 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 are reported.

Amplification of premade plasmid pool

We can amplify premade plasmid library pools 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 then 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.
Alternatively, we can perform NGS validation of your plasmid pool before and/or after transformation. Please note that for plasmid pools 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 via viral transduction, such as infecting with lentivirus or adeno-associated virus. VectorBuilder has developed a range of proprietary technologies and reagents that 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 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 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 a detailed service proposal.