Catalog #: CPLVSGL
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.
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)
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.
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.
On completion, we provide 500 µg of the plasmid library and the following information:
The whole process takes approximately 6 weeks once the gene list is finalized.