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.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE