Custom sgRNA Libraries

Cellecta has the expertise and capability to generate high-quality, complex CRISPR sgRNA libraries targeting virtually any defined sequences and incorporating a range of features for many types of CRISPR-based functional screens.

Our custom library construction team works with you to optimize all aspects of the library for your desired application. We offer a large range of vectors and are often able to customize selection markers, promoters, or other features, as needed. We can also make libraries in non-Cellecta vectors upon request, including AAV and retroviral vectors.

In designing the sgRNA-encoding oligonucleotides, we can easily incorporate unique features required for specialized screening applications. Cellecta's custom sgRNA library service easily accommodates all the variations listed below and more:

• Single-cell applications may use guides with a modified tracrRNA containing feature barcode sequences for capture with 10X Chromium Single Cell 3′ v3 Gel Beads
• Screens with the CRISPRa dCas9-SAM system require an additional stem-loop feature in the sgRNA tracr domains
• 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 Custom CRISPR Library Service ensures production of a quality library that meets your experimental needs.

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 Vector Information) 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.

Resources:

Flyer: CRISPR Libraries for Single-Cell Expression Screening

Flyer: Custom CRISPR sgRNA Pooled Libraries (side 1) and Custom Constructs (side 2)