Abstract
In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.
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Acknowledgment
This work is supported by grants from Guangdong Province Public Interest Research and Capacity Building Special Fund (2015B020201002) and the Ministry of Agriculture of the People’s Republic of China (2016ZX08010-001, 2016ZX08009-002) and the Postdoctoral Science Foundation of China (2016 M602480).
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Xie, X., Ma, X., Liu, YG. (2019). Decoding Sanger Sequencing Chromatograms from CRISPR-Induced Mutations. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_3
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DOI: https://doi.org/10.1007/978-1-4939-8991-1_3
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