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Analysis of Off-Target Mutations in CRISPR-Edited Rice Plants Using Whole-Genome Sequencing

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Rice Genome Engineering and Gene Editing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2238))

Abstract

The CRISPR/Cas systems have become the most widely used tool for genome editing in plants and beyond. However, CRISPR/Cas systems may cause unexpected off-target mutations due to sgRNA recognizing highly homologous DNA sequence elsewhere in the genome. Whole-genome sequencing (WGS) can be used to identify on- and off-target mutation. Here, we describe a pipeline of analyzing WGS data using a series of open source software for analysis of off-target mutations in CRISPR-edited rice plants. In this pipeline, the adapter is trimmed using SKEWER. Then, the cleaned reads are mapped to reference genome by applying BWA. To avoid mapping bias, the GATK is used to realign reads near indels (insertions and deletions) and recalibrate base quality controls. Whole-genome single nucleotide variations (SNVs) and indels are detected by LoFreq*, Mutect2, VarScan2, and Pindel. Last, SNVs and indels are compared with in silico off-target sites using Cas-OFFinder.

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Author information

Authors and Affiliations

  1. Jiangsu Key Laboratory of Crop Genetics and Physiology, Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou, Jiangsu, China

    Guanqing Liu & Tao Zhang

  2. Key Laboratory of Plant Functional Genomics of the Ministry of Education, Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou, Jiangsu, China

    Guanqing Liu & Tao Zhang

  3. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA

    Yiping Qi

  4. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA

    Yiping Qi

Authors
  1. Guanqing Liu
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  2. Yiping Qi
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  3. Tao Zhang
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Corresponding author

Correspondence to Tao Zhang .

Editor information

Editors and Affiliations

  1. Synthetic Biology, Biofuel and Genome Editing R&D, Reliance Industries Ltd, Navi Mumbai, Maharashtra, India

    Anindya Bandyopadhyay

  2. Crop Improvement and Genetics Research Unit, USDA-ARS, Albany, CA, USA

    Roger Thilmony

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Liu, G., Qi, Y., Zhang, T. (2021). Analysis of Off-Target Mutations in CRISPR-Edited Rice Plants Using Whole-Genome Sequencing. In: Bandyopadhyay, A., Thilmony, R. (eds) Rice Genome Engineering and Gene Editing. Methods in Molecular Biology, vol 2238. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1068-8_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1068-8_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1067-1

  • Online ISBN: 978-1-0716-1068-8

  • eBook Packages: Springer Protocols

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