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Custom CRISPR sgRNA Pooled Libraries

In addition to our ready-to-use, off-the-shelf Genome-Wide CRISPR libraries, Cellecta also can generate Custom CRISPR sgRNA Pooled Libraries in as little as 6 weeks.

  • Custom sgRNA libraries for CRISPR knockout, CRISPRa, and CRISPRi
  • Get any guide and tracrRNA sequences desired–standard, HEAT, 10X capture sequences, etc.
  • Cellecta has over 15 years experience making high quality pooled shRNA, barcode, and sgRNA libraries

As shown in the 2014 Science publications of Wang T, et al., and Shalem et al.; pooled libraries of CRISPR sgRNA-expressing constructs can be used to screen thousands of genes throughout a cell population and identify those that are required for a phenotype of interest such as viability, pathway activation, or the appearance of cell surface markers.

Drawing on our experience of over 15 years working with pooled shRNA, barcode, and sgRNA libraries, Cellecta has the expertise and capability to generate high-quality, complex CRISPR sgRNA libraries targeting virtually any defined sequences.

CRISPR/Cas9 diagram showing Cas9 interaction with sgRNA

Screens have been done with variations of the CRISPR system with modified Cas9 proteins that alter the expression level of target genes. Screens can be run where gene targets are selectively inhibited (CRISPRi) or activated (CRISPRa) to assess the effect on phenotypes of interest. Cellecta will also design and provide custom libraries for CRISPRi and CRISPRa screens.

Finally, specialized applications often require sgRNA libraries with specialized designs. For example:

  • Single cell applications may use guides with a modified tracrRNA containing specific sequences (e.g. capture sequences compatible with 10X Chromium Single Cell 3′ v3 Gel Beads)
  • CRISPRa SAM system requires an additional stem loop in the sgRNA,
  • Studies that combine RNA-Seq analysis with CRISPR screens can require an sgRNA cassette that is expressed as part of a transcript barcode,
  • Other types of screens may need to have barcode or unique molecular identifier (UMI) sequences combined with sgRNAs.

Cellecta’s custom sgRNA construct and library service easily accommodates all the above variations and more. Just let us know what you need, and we will provide it.

Cellecta Custom CRISPR Libraries

1. Oligo Design and Synthesis 

For sgRNAs targeting standard human and mouse protein-coding genes, you need only provide us with your list of targets. We design oligonucleotides encoding sgRNAs to your target genes based on 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. For more specialized applications, researchers may also provide their own guide sequences, or we can work with you to design specialized sgRNAs to meet your needs. In addition, we typically employ our improved sgRNA scaffold structure which incorporates the HEAT modifications unless other designs are preferred.

2.  Cloning

After the design step, we synthesize and clone the pool of oligos in any of our standard library vectors (See Resources tab, below) 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 for use when analyzing screening results.

NGS Representation QC of a Cellecta 2K Custom sgRNA Library

3. Quality Analysis

Once the library is made, we isolate a few dozen constructs for full-insert Sanger sequencing to confirm the configuration of the sgRNA expression cassette and ensure correct insertion. We also deeply sequence all guide sequences by NGS, to confirm full representation of the oligo pool, and assess distribution. Libraries that do not meet our standards are remade.

4. Deliverables

On completion, we provide 500 µg of the plasmid library with:

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

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

Optional Packaging: 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.

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

User Manuals

Custom sgRNA Library Vectors

The following CRISPR sgRNA cloning vectors are our standard custom library vectors. If interested in a vector not listed, please see the Vector Information page.

Constitutive Library sgRNA Knockout VectorsNGS Cassette*Sequence**Map
pRSGCCP-U6-(sg)-CMV-Cas9-2A-PuroHTS6C.txt|.gb|.dnaMap

Constitutive Library sgRNA Knockout VectorsNGS Cassette*Sequence**Map
pRSG16-U6-(sg)-UbiC-TagRFP-2A-PuroHTS6C.txt|.gb|.dnaMap
pRSG17-U6-(sg)-UbiC-TagGFP2-2A-PuroHTS6C.txt|.gbk|.dnaMap
pRSGEP-U6-(sg)-EF1-PuroHTS6C.txt|.gb|.dnaMap
pRSGUP-U6-(sg)-UbiC-PuroHTS6C.txt|.gb|.dnaMap

Inducible Library sgRNA Knockout VectorsNGS Cassette*Sequence**MapExample ConstructNGS Amplicon
pRSGT16-U6Tet-(sg)-CMV-TetRep-2A-TagRFP-2A-PuroHTS6CTet.txt|.gb|.dnaMap.dna.dna
pRSGT17-U6Tet-(sg)-CMV-TetRep-2A-TagGFP2-2A-PuroHTS6CTet.txt|.gb|.dnaMapTBA.dna

Safety Data Sheets (SDS)

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