CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

Abstract

Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5' end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites.

Fig 1. CCTop web interface.

(A) Main page containing the input fields to customize the identification of sgRNA target sites and the off-target prediction. (B) Results page providing detailed information of all identified sgRNA target sites.

Fig 2. Experimental verification of sgRNA-1.

(A) In vitro cleavage depending on sgRNA-1/Cas9 occurred on linearized plasmids containing eGFP but not eGFP ^var. Successful cleavage of eGFP plasmid (4128bp) resulted in a 2052bp and 2076bp fragment. The absence of expected fragments (627bp, 4025bp) demonstrated that eGFP ^var (4652bp) was not digested by sgRNA-1/Cas9. (B) A faint double band (2154bp, 2198bp, asterisk) indicated inefficient digestion of off-target 1 (OT#1) while OT#2 and OT#3 (S1 Table) were not cleaved. Note: contrast was enhanced for better visualization. (C) Silent mutations of sgRNA-1 target site in eGFP ^var. (D-E) Injections of sgRNA-1 and Cas9 mRNA into wimb ^-/+ embryos (D) resulted in strong inactivation of eGFP (E).

Fig 3. Visual evaluation of targeting specificity of selected sgRNAs.

Exclusive and homogenous expression of eGFP in the domains of cryaa (A, A’), rx2 (B) and actb (D) was evident already in the injected generation (F0). The integrations were transmitted to the next generation (F1; C, E).