Catalog #: CPLVSGL

Custom sgRNA 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.
  • Libraries can be provided in plasmid and pre-packaged ready-to-transduce formats
  • Multiple vector formats, including inducible and expresion of sgRNA on mRNA for detection in conjunction with RNA-Seq

Cellecta has over 12 years experience making high quality pooled shRNA, barcode, and sgRNA libraries.

Custom sgRNA LibrariesDrawing 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.

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.

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 and the following information:

  • All sequence information on the sgRNA guides and vector
  • Complete cloning site design
  • Primer information for sequencing
  • NGS sgRNA distribution data

The whole process takes approximately 6 weeks once the gene list is finalized.

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