Optimizing CRISPR/Cas9 for Easy Knock Out, Activation or Repression of Genes

Cellecta’s 80K Single-module Human Genome-Wide CRISPR Library was designed to be small enough (~80,000 constructs) to enable convenient genome-wide “dropout viability” screens with most standard human cell modules. With 4 sgRNAs targeting each gene, all of which have been designed using the latest criteria for effective guides described by Doench, et al. and containing the Cellecta-designed HEAT sgRNA structure which we have proven to generate more robust screening results than the standard sgRNA design, the library has deep enough gene coverage to generate efficient results. Genome-Wide Coverage with a Convenient Single 80K Module Single library with ~80,000 constructs targeting approximately 19,000 protein-coding genes Each gene is targeted by 4 sgRNAs designed based on Doench, et al. criteria Targets all human protein-coding genes—over 19,000 gene targets. The 80K Single-Module Human Genome-Wide CRISPR Library includes the following controls standard non-targeting controls non-specific control (sgRNA targeting introns throughout the genome) lethal sgRNA targeting generally essential genes (i.e., positive controls for dropout screens)

Want to know if Cas9 is active in your cells? Cellecta’s CRISPRtest™ method allows you to assess the level of Cas9 activity in your cell line using two pre-mixed lentiviral-packaged vectors, the first containing a green fluorescent protein (GFP) marker and gRNA sequence targeting an essential gene (PCNA), and the second containing a red fluorescent protein (RFP) marker and a non-targeting gRNA. Transduction of CRISPRtest virus into cells will result in a mixture of cells expressing GFP and cells expressing RFP. Upon cell growth in the presence of active Cas9, the ratio of GFP cells: RFP cells will decrease proportionally to Cas9 activity. The CRISPRtest Functional Cas9 Activity Kit, currently for human cell lines only, is an easy-to-use, highly sensitive kit for measuring functional Cas9 activity in almost any human cell line.