Library Construction

VectorBuilder can help you build custom pooled libraries to perform large-scale functional screens. We have extensive experience in constructing high-quality pooled libraries utilizing our optimized vectors and proprietary technologies. We can deliver your library as E. coli stock, DNA, or recombinant virus, depending on your needs. Our custom libraries are fully validated by next-generation sequencing (NGS) so you know exactly what you get.

Types of custom libraries we can construct:
  • CRISPR libraries (e.g. CRISPR KO, CRISPRa/i, CROP-seq, Perturb-seq, etc.)
  • shRNA libraries
  • Barcode libraries
  • Enhancer/promoter screening libraries
  • Peptide expression libraries
  • DNA shuffling libraries
  • Two-hybrid libraries
  • Capsid screening libraries
  • And many more

Service Details

Price and turnaround
Service Brief Description Price (USD) Turnaround
Design library construction strategy* Free 1-4 days
Pooled library construction Includes oligo/gene synthesis, massive parallel cloning of inserts for variable region into desired vector backbone, and preliminary validation of the plasmid pool by Sanger sequencing.** From $2,000 3-5 weeks
Amplification of premade plasmid pool**** If you have a premade library in the form of 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 $139 4-6 days
Virus packaging of pooled library Please click here to view detailed scales of virus packaging services. ***
NGS validation of premade pooled library**** We can validate the quality of your premade pooled library 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 $200 per sample 5-7 weeks

* This free service is limited to cloning strategy design. For designing the sequences for the variable region, such as gRNA and shRNA, there may be additional charge and turnaround.

** The default deliverable is E. coli glycerol stock.

*** The price for packaging library plasmid is 1.5-fold of the price for packaging single vector plasmid.

**** For pooled library not constructed by VectorBuilder, we cannot guarantee the complexity or uniformity of the library because we have no control over the composition of your premade library.

Technical Information

Genetic screens mediated by pooled CRISPR libraries and shRNA librariesView more

Pooled CRISPR libraries

Pooled CRISPR libraries are powerful tools 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:

Screening Strategy Mechanism Target Location Application
CRISPR KO 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 Catalytically inactive Cas9 (dCas9) fused to a transcriptional activator (e.g. VP64) is used to activate genes regulated by target sites. Usually promoter regions and other noncoding regulatory regions Gain-of-function screens of coding genes, or screens for regulatory function of noncoding regions
CRISPRi Catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor (e.g. KRAB) is used to inhibit genes regulated by target sites. Usually promoter regions and other noncoding regulatory regions Loss-of-function screens of coding genes, or screens for regulatory function of noncoding regions

In addition to custom pooled CRISPR library construction, VectorBuilder offers high-quality premade dual-gRNA libraries targeting human and mouse genes. 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. These libraries have been fully validated by next-generation sequencing and functional assays.

Click to view more information of our premade dual-gRNA libraries

Pooled shRNA libraries

Pooled shRNA libraries can serve as powerful and cost-efficient tools 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.

Click to view more information of our premade pooled shRNA libraries 
Pooled library construction workflowView more

Design library construction strategy

If you need assistance in designing your gRNA or shRNA sequences for your list of target genes or genomic regions, VectorBuilder 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.

Pooled library construction

VectorBuilder uses 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 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 plasmid pool, with sequencing depth at >500x coverage. NGS data is analyzed and all designed variable region sequences with normalized abundance in the pool are reported.

Amplification of premade plasmid pool

VectorBuilder 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 library coverage.

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.

Click to view more information of virus packaging services 

NGS deconvolution of post-screening sample

VectorBuilder can help you identify hits by NGS for your CRISPR or shRNA screens. 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 or 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.

How to Order

Customer-supplied library plasmid pool

If the customer-supplied premade plasmid pools are used, please send us the materials 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 pools, we cannot provide any guarantees regarding the complexity or uniformity of the library.

FAQ

Should I use single gRNA or dual gRNA for CRISPR-mediated knockout?

For CRISPR-mediated genome editing, Cas9 nuclease is directed to the target site of site-specific guide RNA (gRNA) in the genome to create DNA cleavage. In most cases, to generate simple gene knockout, a single gRNA can be used together with Cas9 to generate a double-strand break (DSB), which is then inefficiently repaired by the non-homologous end joining (NHEJ), resulting in permanent mutations, such as small insertions or deletions, at the site of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc.

Dual gRNAs can be used if Cas9_D10A nickase is being used to target the two opposite strands of a single target site. In this approach, the nickase enzyme will generate single strand cuts on both strands, one guided by each of the two gRNAs, resulting in DSBs at the target site. Generally, this method reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.

Dual gRNAs can also be used when Cas9_D10A nickase and an exogenous donor DNA template are being used to introduce specific base-changes (e.g. knockins) into a gene of interest. In this approach, the two opposite strands would be targeted by the two gRNAs at two sites flanking the desired mutation site, and homology-directed repair (HDR) pathways make use of the exogenous donor template to repair the excised sequence.

Click to view options of our dual gRNA vectors 
For CRISPR knockout screens, should I use 1-vector systems or 2-vector systems?

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.