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.
Inhibition of The Auto-inflammation Suppressor Protein ISG15 Triggers Preeclampsia by Blocking Trophoblast Migration and InvasionPoster
In summary, ISG15 expression levels are crucial for trophoblast morphology and function (migration/invasion). By blocking trophoblast invasion, reduced ISG15 levels could contribute to impaired spiral artery transformation that reduces utero-placental blood flow in preeclampsia. Thus, agents inducing ISG15 expression are likely to be therapeutic in preeclampsia.
An Emerging Phenotype of Partial RAG 1/2 Deficiency Among Young Children with Autoimmunity and Viral InfectionsPoster
We describe the natural history of a cohort of 12 patients with confirmed partial RAG1/2 mutations and autoimmunity at a young age. We were seeking the link between viral infections and autoimmunity and tested candidate biomarkers that may reflect the underlying RAG1/2 protein deficiency.READ MORE
Severe Combined Immunodeficiency (SCID) caused by thymic aplasia due to a mutation in TBX1Poster
We report one of the first cases of TBX1 haploinsufficiency causing complete thymic aplasia and SCID.READ MORE
Comments | 0 ADD COMMENT
Precision Medicine & Biomarkers Leaders Summit: USA
May 07 - May 08, 2018