Functional characterization studies of the human genome have delivered a tremendous amount of information through using genome-based loss-of-function screening in diverse models. RNA interference (RNAi) has been used as the predominant method for loss-of-function of genome screening, but it is limited by variable efficiency, frequent incompleteness of protein depletion and confounding off-target effects. CRISPR/Cas9 technology is ideally suited for genome‐wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. However, certain types of mammalian cells are difficult to transfect using lipid reagents or electroporation. In order to circumvent these difficulties, lentiviral vectors are commonly used as another delivery method as they can be easily titrated to manipulate transgene copy number and are stably maintained by integration into the genomic DNA during subsequent cell replication.
Figure 1. An experimental flowchart for using pooled lentiviral CRISPR libraries.
As one of the pioneers in creating libraries, Creative Biogene has developed a proficiency in producing high-quality libraries expressing short genetic elements, such as peptides, shRNA, enzyme active site elements, and sgRNA. Our QVirus™ platform can offer the packaging of your CRISPR libraries into our lentiviral vectors. CRISPR lentiviral libraries are packaged into highly pure and concentrated lentiviral particles, either from an existing lentiviral plasmid library or from a custom plasmid library that we construct starting from a provided oligonucleotide pool, corresponding to your guide sequences.
QVirus™ Platform can provide scientists with lentiviral vectors libraries in arrayed or pooled format for low- or high-throughput screening. We will assist you in technical choices related to lentiviral particles.
|Format||Volume||Concentration||Number of Guide||Application|
|Arrayed||200 ul||1x10^6 TU/ml(crude harvest)||Up to 2500||For permissive immortalized cell lines only|
|Pooled||1-30 ml||1x10^7 or 1x10^9 TU/ml (flexible concentration/purification)||Theoretically no limit||For immortalized, primary and stem cells|
The CRISPR lentiviral libraries in an arrayed format are ideal to identify the function of hundreds of genes inside a subfamily or a specific pathway. We manufacture customized lentiviral vector batches, carrying your CRISPR lentiviral libraries in arrayed format for the low-throughput screening of permissive immortalized cell lines only. The advantages of arrayed format lentiviral libraries include:
(1) Powerful high-throughput loss-of-function screening tools for target identification;
(2) The gRNA libraries targeting various human gene sets, such as GPCR and kinases are available as pre-made, ready to use lentiviral particles which are arrayed in 96-well plate format;
(3) Contain four sequence-verified distinct gRNA constructs per gene per well;
(4) The average titer of lentiviral CRISPR library particles is >106 TU/ml.
A pooled lentiviral screen can be performed to identify genes that regulate cellular responses and signaling pathways, or to discover novel gene functions. Pooled screening libraries can consist of over 50 constructs up to many thousands. Moreover, pooled screening libraries allow the researcher to transduce and screen a population of cells within a few tissue culture dishes. The features of pooled format lentiviral libraries include:
(1) High-quality pools provided as purified, concentrated lentiviral particles;
(2) Provided as gene families or whole-genome libraries;
(3) Customizable promoter and reporter options, including inducible promoters;
(4) Custom mouse, rat, and human libraries available;
(5) Complete workflow planning tools and protocols
If you have any special requirements, please feel free to contact us. We are looking forward to working together with your attractive projects.
1. Mcdade J R, et al. Practical Considerations for Using Pooled Lentiviral CRISPR Libraries: Considerations for Pooled Lentiviral CRISPR Libraries. Current Protocols in Molecular Biology. John Wiley & Sons, Inc. 2016.