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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References
Cho SW, Kim S, Kim JM et al (2012) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232
Yu C, Zhang Y, Yao S et al (2014) A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in Zebrafish. PLoS One 9:e98282
Nekrasov V, Staskawicz B, Weigel D et al (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693
Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Zischewskia J, Fischer R, Bortesi L (2017) Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnol Adv 35:95–104
Xue LJ, Tsai CJ (2015) AGEseq: analysis of genome editing by sequencing. Mol Plant 8:1428–1430
Lu Y, Ye X, Guo R et al (2017) Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system. Mol Plant 10:1242–1245
Ma X, Zhang Q, Zhu Q et al (2015) A robust CRISPR/Cas9 system for convenient high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284
Ma X, Chen L, Zhu Q et al (2015) Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products. Mol Plant 8:1285–1287
Liu W, Xie X, Ma X et al (2015) DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol Plant 8:1431–1433
Xie X, Ma X, Zhu Q et al (2017) CRISPR-GE: a convenient software toolkit for CRISPR-based genome editing. Mol Plant 10:1246–1249
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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