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.
Highlights
A single guide RNA provides specificity and targets the CRISPR associated 9 (Cas9) endonuclease to introduce a double strand break (DSB) at the site determined by the sgRNA [3]
The plasmids were pre-linearized with BsaI (NEB) about 2kb from the sgRNA target site. 3nM of the pre-linearized plasmids were incubated for one 1h with 30nM sgRNA-1 and 30nM Cas9 protein (NEB) and supplemented with Cas9 nuclease buffer (NEB) in a 30μl reaction volume at 37°C
From any provided DNA sequence all sgRNA target sites will be identified according to adjustable parameters like the type of protospacer adjacent motif (PAM) (‘NGG’ or ‘NRG’), the identity of the two most 5’ nucleotides (‘NN’, ‘GN’ or ‘GG’) [8,25] as well as the two most 3’ nucleotides (‘NN’ or ‘GG’) [26]
Summary
Targeted genome editing has become available to literally all (model) organisms with the emergence of engineered nucleases like transcription activator-like effector nucleases (TALEN) [1], Zinc-finger nucleases (ZFN) [2] or the RNA guided nucleases [3] facilitating the introduction of a double strand break (DSB) at any locus of choice [4,5,6,7,8,9]. Since a stretch of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas system [3,13,14,15], an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. To this end, already a number of online sgRNA target finding and evaluation tools like CRISPR Design [13], E-CRISP [16] or CHOPCHOP [17] have been presented. We present CCTop as an experimentally validated system for the rapid selection of high quality target sites for gene inactivation, non-homologous end-joining as well as homology directed repair
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