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Cellecta received a Phase I NIH SBIR Grant to develop CRISPR guide RNAs (gRNAs or sgRNAs) for human and mouse genes and then use these to build a knockout library targeting 6,500 key disease-related genes in major signal transduction pathways.

Targeting a gene for knockout using CRISPR requires a short strand of RNA (a “guide” strand) that contains a gene targeting region complementary to part of the target gene sequence, and a binding region for the Cas9 nuclease. While it is generally known how to design sgRNAs, the particular elements and features necessary for effective and specific sgRNA strands has not been well defined. Variations of the targeting region and Cas9 binding domain of sgRNAs, such as the ones below that we tested in individual constructs, have been shown to alter the efficacy of sgRNAs.

In this first phase of the grant, Cellecta plans to test a number of sgRNA structure modifications that have been reported to increase Cas9-sgRNA activity and/or specificity. We will use our expertise in Construction and screening of pooled lentiviral libraries large complex pooled sgRNA libraries to simultaneously analyze the efficiency and specificity of a dozen different sgRNA structures to large numbers of essential targets. The libraries will also include controls to non-essential genes, as well as non-targeting sgRNAs.

Screens using these libraries made of different sgRNA structures to the same set of targets will provide sufficient examples with each structural variant for robust statistical analysis so that the effects of each variation can be reliable assessed. After completing this testing, we will use the most effective sgRNA in designing a CRISPR library targeting key disease-related genes in major signal transduction pathways.

gRNA-tracrRNA-design-comparison-6days

Figure 1. The efficiency of GFP knockout on three gRNAs changes when the complementary targeting sequence is shortened or alternations are made to the Cas9-binding domain (“AT” and “HE”). The variations tested above were described by Chen, et al. (Cell 2013, 155:1479-91) and Fu, et al (Nat. Biotechnol. 2014, 23:279-84).