Design Considerations for Highly Specific and Efficient Synthetic crRNA Molecules
Poster Oct 12, 2015
Anja van Brabant Smith, Emily M. Anderson, Shawn McClelland, Elena Maksimova, Tyler Reed, Steve Lenger, Žaklina Strezoska, Hidevaldo Machado Dharmacon, part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, US
In order to understand the parameters affecting CRISPR-Cas9 gene editing efficiency, we systematically transfected synthetic CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) reagents targeting components of the proteasome into a reporter cell line in which knockout of proteasome function results in fluorescence of a ubiquitin-EGFP fusion protein that is normally degraded by the proteasome pathway. We evaluated the functionality of > 1100 crRNA sequences in this system; using these data, we developed and trained an algorithm to score crRNAs based on how likely they are to produce functional knockout of targeted genes. We further tested our algorithm by designing synthetic crRNAs to genes unrelated to the proteasome and examined their ability to knock out gene function using additional phenotypic assays. To augment our functionality algorithm, we developed an optimized alignment program to perform rapid, flexible, and complete specificity analysis of crRNAs, including detection of gapped alignments. We have combined this comprehensive specificity check with our functionality algorithm to select and score highly specific and functional crRNAs for any given gene target.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE