Complete alignment identification of CRISPR-Cas9 genomic off-targets using Edit-R CRISPR specificity tool and a comprehensive analysis of positional mismatch tolerance
Poster Jun 23, 2017
Emily M. Anderson, Shawn McClelland, Amanda Haupt, Eldon T. Chou, John Schiel, Annaleen Vermeulen, Žaklina Strezoska, Hidevaldo Machado, Steve Lenger, Amanda Birmingham, and Anja van Brabant Smith Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, USA
The CRISPR-Cas9 system has the potential to advance basic and applied research; however, specificity of RNA-directed DNA cleavage events is not yet completely understood and can hamper its wider application. New findings on off-target effects and their determinants are being reported frequently. Recent work has demonstrated gene editing by CRISPR RNAs (crRNAs) containing bulges of up to 4 nucleotides, but existing design tools are unable to detect putative off-targets based on gapped alignments. We present the Dharmacon™ Edit-R™ CRISPR specificity tool, a simple web tool that leverages well-characterized alignment optimization techniques to perform rapid, customizable, and complete crRNA specificity checking including gap detection. The Edit-R CRISPR specificity tool is freely accessible dharmacon.gelifesciences.com/tools-and-calculators/crispr-specificity-tool In addition, we have comprehensively evaluated positional off-targeting propensities of the CRISPR system using a three-component platform of Cas9, synthetic crRNAs, and synthetic tracrRNA. We executed a systematic positional screen of crRNAs containing two nucleotide mismatches to the DNA target for two functional crRNAs. The application of a high-throughput reporter assay that directly measures functional activity of a central cellular process (ubiquitin-proteasome activity), allowed measurement of the relative cleavage activity of all disruptive two-mismatch combinations for each crRNA (190 combinations per sequence or 380 total), regardless of the need for the off-target target region to naturally occur in the genome adjacent to a PAM. Our results demonstrate that while off-targeting does occur in the presence of two mismatches between the crRNA and the target DNA, the overall levels of functional off-targeting are low relative to on-target activity. Analysis of the position of tolerated mismatches further clarifies the mechanism of CRISPR-Cas9 off-targeting as well as provides additional crRNA design rules for mammalian gene editing. These studies demonstrate the simplicity and high-throughput nature of this three-component system for elucidation of CRISPR-Cas9 function and mechanism.
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
The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs.READ MORE
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.