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Custom CRISPR sgRNA Pooled Libraries

  

In addition to our ready-to-use, off-the-shelf Genome-Wide CRISPR libraries, Cellecta offers a custom CRISPR library construction service. 

CRISPR technology exploits a bacterial system that slices up DNA from invading viruses to specifically target and permanently disrupt (“knock out”) genes in mammalian cells. Targeting a gene for knockout using CRISPR simply requires a short strand of RNA (sgRNA) that contains a region matching part of the gene sequence of interest, and the Cas9 nuclease protein. As a result, sgRNA-expressing constructs can be made to target and knock out essentially any gene, and as shown in the 2014 Science publications by Wang T, et al., and Shalem et al., collections of these sgRNA constructs interrupt expression of thousands of genes throughout a cell population. The cells can then be screened to look for the appearance or elimination of various phenotypes, such as viability, pathway activation, or the appearance of cell surface markers.

Pooled lentiviral-based libraries containing heterogeneous mixtures of CRISPR sgRNAs (or gRNA) constructs allow you to assay the effects of many thousands of knockouts in one experiment. Drawing on our experience of over 10 years working with pooled shRNA libraries, Cellecta has the expertise and capability to generate high-quality, complex CRISPR sgRNA libraries targeting virtually any defined sequences.

In addition, screens have been done with variations of the CRISPR system with modified Cas9 proteins that, rather than disrupt the genomic DNA and completely knock out the target gene, instead affect expression of the target gene. With these modified CRISPR systems, screens can be run where gene targets are selectively inhibited (CRISPRi) or activated (CRISPRa) to assess the effect on phenotypes of interest. The libraries for CRISPRa and CRISPRi screens require similar sgRNA structures, but with a design that targets the promoter regions. In addition to ready-to-use, genome-wide CRISPRi and CRISPRa libraries, Cellecta also provides custom libraries for CRISPRi and CRISPRa screens.

 

Process to Make Custom CRISPR Libraries

You provide us with your list of targets, and Cellecta does the rest!

We design oligonucleotides encoding sgRNAs to your target genes. We incorporate the latest guidelines from published literature (e.g. Doench, et al., Nature 2016) as well as some other features we have optimized based on our screening work, to maximize effectiveness and minimize off-target activity. In addition, we also use our improved sgRNA scaffold structure which incorporates the HEAT modifications to the non-targeting tracr sequence on the sgRNA molecule.

After the design step, we synthesize and clone the pool of oligos in any of our standard vectors or, in many cases, we can make the library in a customer-provided vector. We can also include a panel of non-targeting, intron-targeting, non-specific cutting, and lethal sgRNA controls to provide reference standards when analyzing screening results.

Once the library is made, we isolate a few dozen constructs for full insert sequencing to confirm the configuration of the sgRNA expression cassette and ensure correct insertion; then we also deeply sequence all guides by NGS, to confirm full representation fo the oligo pool, and assess distribution. Libraries that don’t meet our standards are remade.

On completion, we provide 500 ug of the plasmid library with

  • all sequence information on the sgRNA guides and vector
  • the cloning site design
  • primer information for sequencing
  • NGS distribution data

We also provide optional packaging services for lentiviral-based libraries so you can receive VSV-g pseudotyped viral particles ready to introduce onto cells for a screen.

The whole process takes approximately two months once the gene list is finalized.

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 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 sgRNA sequences
  • Performs NGS on the Illumina NextSeq or HiSeq
  • Enumerates 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