CT-Finder: A Web Service for CRISPR Optimal Target Prediction and Visualization.

Houxiang Zhu,Lauren Misel,Chun Liang,Michael L. Robinson,Mitchell Graham

doi:10.1038/srep25516

Abstract

The CRISPR system holds much promise for successful genome engineering, but therapeutic, industrial, and research applications will place high demand on improving the specificity and efficiency of this tool. CT-Finder (http://bioinfolab.miamioh.edu/ct-finder) is a web service to help users design guide RNAs (gRNAs) optimized for specificity. CT-Finder accommodates the original single-gRNA Cas9 system and two specificity-enhancing paired-gRNA systems: Cas9 D10A nickases (Cas9n) and dimeric RNA-guided FokI nucleases (RFNs). Optimal target candidates can be chosen based on the minimization of predicted off-target effects. Graphical visualization of on-target and off-target sites in the genome is provided for target validation. Major model organisms are covered by this web service.

Highlights

Cas nucleases, derived from bacterial adaptive immune systems, possess the ability to cause double-strand breaks at targeted locations in genomes
Even with a guide RNAs (gRNAs) sequence that is entirely unique within the genome of interest, Cas nucleases may still cleave at unintended off-target sites that bear very high similarity to the target DNA sequence
As an improvement over the Cas[9] system, the Cas[9] D10A nickases (Cas9n) system operates on the biological principle that two single-strand breaks in close proximity are equivocal to a double-strand break

Summary

Introduction

Cas nucleases, derived from bacterial adaptive immune systems, possess the ability to cause double-strand breaks at targeted locations in genomes. Unlike transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) – tools for sophisticated genomic editing – the CRISPR/Cas system is highly adaptable and programmed This is because CRISPR/Cas eliminates the need for protein engineering, and in turn only requires the design of a simple and short (~20 nt) guide RNA (gRNA) that base pairs with the intended on-target genomic site[2]. The stringency of the requirements for RFNs results in acutely specific targeting, essentially eliminating Cas9-induced off-target mutagenesis, but comes at the disadvantage of reduced target space and narrower applicability Both Cas9n and RFNs systems utilize paired gRNAs and claim high specificity of gene editing due to the effective. Doubling of the number of base pairs matched to the target sites, but have a more limited set of possible target candidates as compared to Cas[9]

Methods

Discussion

Conclusion