Abstract
The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM (available at https://doi.org/www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing (NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers, cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.
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Acknowledgements
We are extremely grateful to Ruiqiang Li from Novogene Bioinformatics Institute for critical reading of the manuscript. We thank Zheng Ruan and his team from Novogene Co., Ltd for NGS technical service. We also thank Yangwen Qian from Hangzhou Biogle Co., Ltd for rice transformation. This work was supported by the National Key Research and Development Program of China (2017YFD0102002), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and the National Natural Science Foundation of China (31401363).
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Liu, Q., Wang, C., Jiao, X. et al. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci. China Life Sci. 62, 1–7 (2019). https://doi.org/10.1007/s11427-018-9402-9
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DOI: https://doi.org/10.1007/s11427-018-9402-9