Rapid Decoding of Sequence-Specific Nuclease-Induced Heterozygous and Biallelic Mutations by Direct Sequencing of PCR Products

Journal Article / 2015

Ma, Xingliang; Chen, Letian; Zhu, Qinlong; Chen, Yuanling; Liu, Yao-Guang

The recent development of sequence-specific nuclease systems, i.e., TALENs and CRISPR/Cas9, has made genomic targeting easier in many organisms including plants (Li et al., 2012; Cong et al., 2013; Joung and Sander, 2013; Li, et al., 2013; Shan et al., 2013; Liang et al., 2014; Zhang et al., 2014). Mutations induced by CRISPR/Cas9 usually occur around the cleavage sites at three bases upstream of the protospacer-adjacent motif (PAM), producing insertion and deletion of nucleotides. For diploid organisms, such targeted mutations may happen in one or both homologous chromosomes. Previous reports showed that CRISPR/ Cas9-based genomic editing in some plants mainly produced complicated mosaic (chimeric) mutations in the somatic cells of the first generation transgenic plants (Li et al., 2013; Mao et al., 2013), and the presence of targeted mutations could be detected by a combination of Cas9 protein and in vitro produced single guide RNAs (sgRNAs) (Gao and Zhao, 2014). However, genomic targeting (Zhang et al., 2014) and our results (Ma X et al., unpublished results) in rice T0 plants show that the majority of targeted mutations are in uniform allelic statuses, mostly biallelic (two distinct variations), homozygous (two identical mutations), and heterozygous (wild-type/single mutation), and many targeted mutations in Arabidopsis T1 plants using our CRISPR/Cas9 system are simple heterozygous and biallelic mutations. Most of the targeted mutations are 1-bp insertions and small segment deletions; nucleotide substitutions and insertions of 2 bp or more are very rare. Direct sequencing of PCR products containing such heterozygous and biallelic mutations results in superimposed sequencing chromatograms. A commercial sequence analysis software package, CodonCode Aligner (http:// www.codoncode.com/), has been developed to decode heterozygous DNA sequences by splitting the overlapping sequencing traces into pseudo-alleles. However, this decoding program is very sensitive to the quality of sequencing chromatograms and often outputs false results. For example, we tested decoding of 21 sequencing chromatograms with heterozygous and biallelic mutations from T0 rice plants using CodonCode Aligner but 16 cases produced false results; four cases of the decoding are shown in Supplemental Figure 1. Therefore, PCR products containing non-homozygous mutations need to be cloned and multiple clones for each targeted site are sequenced, which is tedious, inefficient, and expensive. Here, we present a simpleandhighly reliablemethod for rapiddecodingof suchsuperimposed sequencing chromatograms from direct sequencing of PCR products with heterozygous and biallelic mutations.