Stemmer Manuel, Thumberger Thomas, Del Sol Keyer Maria, Wittbrodt Joachim, Mateo Juan L
Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany.
PLoS One. 2015 Apr 24;10(4):e0124633. doi: 10.1371/journal.pone.0124633. eCollection 2015.
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.
CRISPR/Cas9系统的工程改造为定点诱变和靶向基因组修饰带来了大量新机遇。其中的关键在于引导RNA（gRNA）5'端的一段20个核苷酸的序列，它为结合的Cas9核酸内切酶提供特异性。由于20个核苷酸的序列在给定基因组中可能会多次出现，并且CRISPR/Cas9复合物似乎能接受一些错配，因此在计算机上对靶向位点进行高效且可靠的选择和评估是实验成功的关键前提。在此，我们推出CRISPR/Cas9靶点在线预测工具（CCTop，http://crispr.cos.uni-heidelberg.de），以克服现有工具的局限性。CCTop提供了一个直观的用户界面，带有合理的默认参数，用户可轻松调整。根据给定的查询序列，CCTop会根据脱靶质量对所有候选sgRNA靶向位点进行识别和排序，并显示完整的文档。CCTop已通过基因失活、非同源末端连接以及同源定向修复的实验验证。因此，CCTop为实验生物学家提供了一种快速高效识别高质量靶向位点的工具。
