There are many technical challenges that need to be overcome to produce sgRNA libraries with representative, evenly distributed elements that are amenable to Next-Generation Sequencing (NGS) read-outs.
Our well experienced partner Cellecta offers both, ready-to-use, off-the-shelf Genome-Wide CRISPR libraries, as well as custom-designed CRISPR sgRNA Pooled Libraries targeting virtually any set of genes. You can choose from a large range of vectors with a variety of selection markers, promoters, or other features, as needed.
Nucleus Biotech works with you to optimize all aspects of the library for your desired application.
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 (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 typically employ our improved sgRNA scaffold structure which incorporates the HEAT modifications unless other designs are preferred.
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
After the design step, we synthesize and clone the pool of oligos in any of our standard library vectors (See Vector Information) or 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.
We provide 500 µg of the plasmid library and the following information:
– All sequence information on the sgRNA guides and vector