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Custom CRISPR and RNAi Pooled Libraries

 

 shRNA and CRISPR Pooled Library Construction workflow

Construction of shRNA and CRISPR Libraries

By taking advantage of array-based oligonucleotide synthesis, we can readily make precisely defined large custom pooled libraries expressing many thousands of elements. Within approximately 3 months, we can produce a completely new, high quality custom library. The library vector can also be easily customized, and the same library oligonucleotides can be cloned into more than one vector backbone (e.g. with an inducible and constitutive shRNA or sgRNA promoter). It is a very flexible platform.

 

Cellecta has the expertise and capability to generate high quality complex heterogeneous libraries consisting of virtually any defined sequences. This capability combined with our proprietary vectors and lentiviral expertise provides Cellecta with the ideal platform to create excellent quality, highly representative pooled genome-wide and targeted lentiviral sgRNA CRISPR Custom libraries, shRNA Custom libraries for RNAi screens and Libraries for Barcoding Cells.

 

Cellecta Libraries feature

 

  • A higher percentage of functional shRNAs, designed using validated shRNA sequences and optimized shRNA structures
  • Flexible array-based oligonucleotide synthesis enables rapid creation of complex libraries expressing any set of shRNAs and sgRNAs to any targets
  • Optimized shRNA and sgRNA expression vectors with various markers, selections, and both inducible and constitutive promoters
  • Lentiviral-based system ensures efficient delivery of high complexity, pooled custom shRNA and CRISPR libraries into a wide range of cell types
  • Compatible with Illumina HiSeq, GAIIx, and NextSeq NGS platforms

   

For more information on how Cellecta builds our pooled shRNA and sgRNA expression libraries, please see our Technology section.

 How Many shRNAs or sgRNAs Do You Recommend Per Transcript? How Effective is the Knockdown/ Knockout?

How Many shRNAs or sgRNAs Do You Recommend Per Transcript? How Effective is the Knockdown/ Knockout?

 

For broad coverage, we usually design libraries so there are at least 5-6 shRNAs or sgRNAs targeting a specific transcript. Our approach to shRNA design draws on in-house design and structural studies. We can design libraries to target each transcript with as many shRNAs or sgRNAs as desired. Some labs are interested in designing shRNA or sgRNA to “tile” target transcripts. We have optimized the shRNA structure (e.g., length, loop size, mismatches, etc.) and sgRNA structure so that they produce highly effective target knockdown or knockout and are amplified and maintained effectively in a pooled library. On average, more than 65% of the shRNAs knock down transcript levels by greater than 70%. More than 90% of cells expressing Cas9 and sgRNA knock out transcript levels after about 9 days of expression.

What Sizes of Custom shRNA and CRISPR Libraries Do You Offer?

 

We provide custom-designed shRNA or sgRNA libraries targeting any set of genes of interest. Since we utilize array-based synthesis for library inserts, the oligonucleotide pool sizes are somewhat fixed.

 

  • Typical library sizes range anywhere from 1,000 to 80,000 shRNAs
  • Targeting up to genome-wide coverage
  • For specialized applications such as in vivo screening, we can provide small libraries (targeting <1,000 transcripts or genes)

Custom Library Service

 

The library construction service includes oligo design, oligo synthesis, library construction, and QC by clone sequencing and NGS. Deliverables include 500 μg plasmid DNA library, the plasmid cloning vector, library sequences, clone sequencing QC data, and representational analysis with NGS data.

 

  • shRNA library prices start from $25,000
  • CRISPR sgRNA library prices start from $21,000

 

Cellecta can package the libraries and provide them in ready-to-transduce pseudoviral format at additional cost. Packaging of the Cas9 construct for CRISPR libraries can also be provided.

Vector Design for Custom shRNA and CRISPR Libraries

  

Standard library expression vector with example markers and promotersOur standard library vector expresses shRNA or sgRNA from a constitutive U6 promoter and contains puromycin antibiotic selection and a red fluorescent protein marker (as shown). However, as indicated in the figure, we can provide customized changes to any of these elements, including using an inducible promoter. We find that, for library screening in cell culture, inducibility adds an additional variable that can complicate the assay unnecessarily (more information about this is available in our blog entry, “RNAi Screening with An Inducible Promoter: Is There an Advantage?”). However, for specialized applications, we can certainly provide libraries with these inducible H1 or U6 promoters upon request.

 

  

For more information on loss-of-function screening, visit the Library Technology page.

 

If you are interested in off-the-shelf, whole human genome CRISPR or shRNA or mouse shRHNA pooled lentiviral libraries, please visit our Products page.

 

User Manuals

shRNA Libraries

  • Custom Pooled Lentiviral shRNA Libraries, v4 (PDF)
  • Pooled Lentiviral shRNA Library Screening Reference Manual, v2a (PDF)

CRISPR sgRNA Libraries

  • Custom and Premade Pooled Lentiviral CRISPR sgRNA Libraries, v2 (PDF)

Next-Gen Sequencing of Samples from Genetic Screens

Cellecta shRNA and CRISPR libraries are provided with a complete protocol and all sequencing information to enable researchers to perform high throughput genetics screens and analysis. However, we do also provide Next-Gen Sequencing (NGS) and analysis services for researchers running their own screens with our libraries. Just provide harvested cells for each time point or treatment condition (one sample), and we extract DNA, amplify, sequence, and assemble the data with some basic analysis.

You provide Cellecta with frozen cell pellets or tissue after screening…

…and Cellecta does the rest:

  • Extracts genomic DNA
  • Amplifies barcodes or sgRNA sequences
  • Performs NGS on the Illumina NextSeq or HiSeq
  • Enumerates shRNA or sgRNA counts from raw sequencing data

Please see the Next-Gen Sequencing and Analysis web page for additional information and how to order.

Custom CRISPR sgRNA Libraries

Custom shRNA Libraries

Please email sales@cellecta.com to obtain a quotation.

Related Products:

Catalog #Item DescriptionPrice
SVC9-PSpR-CMV-Cas9-2A-Hygro Cas9 Expression Vector (plasmid), 25 μg$300
SVC9-VSpR-CMV-Cas9-2A-Hygro Cas9 Expression Vector (virus), 1 x 106 TU$500
SVC9B-PSpR-CMV-Cas9-2A-Blast Cas9 Expression Vector (plasmid), 25 μg$300
SVC9B-VSpR-CMV-Cas9-2A-Blast Cas9 Expression Vector (virus), 1 x 106 TU$500
CPCP-K2AReady-to-Use Lentiviral Packaging Plasmid Mix, 250 μg
(for (25) 10-cm plates)
$325
LFVC1LentiFuge™ Viral Concentration Reagent, 1 ml
(for 1 liter supernatant)
$325
CRTESTCRISPRtest™ Functional Cas9 Activity Kit (Human)
(for 5 cell line pairs)
$750