Efficient Identification Of Targeted Genome Modification In Plant Using Single-strand Conformation Polymorphism Assay

Targeted gene editing can induce indel mutations by several of sequence specific nucleic acid enzymes such as the latest CRISPR-Cas9, previous zinc-finger nucleases and transcription activator-like effecter nucleases. This is produced by efficient DNA double strand breaks at the target genomic sequence and then repaird by the error-prone nonhomologous end joining DNA repair system or the precisely homology directed repair. So far, the dominant approach for genotyping of genome-modified plants or animals is using SURVEYOR nuclease, Cel-I or T7E1 to recognize and cut the mismatched sites of hetero-duplexes. The restriction enzyme assay and high resolution melting curve analysis are also common methods to detect the efficiency of the induced mutations.However, current assays have lots of limitations. They either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. So looking for a more appropriate directional modification site detection method is very important. In this study, in order to get a better detection means, the traditional SSCP method is applied to the detection of gene modification.Single-strand conformational polymorphism(SSCP) analysis is a simple and sensitive technique for mutation detection and genotyping. It is based on the defined conformation that single-stranded DNA has. A single base change in the sequence can alter the conformation which cause single-stranded DNA to migrate differently under native polyacrylamide electrophoresis conditions. So we can distinguish the wild-type and mutant DNA samples via different band patterns.In this study, we established a method based on the SSCP analysis system by optimizing the main experimental conditions including gel concentration, gel ratio, electrophoresis voltage and time to detect the plant genome modifications induced by sequence specific nucleic acid enzymes. The repeatability, sensitivity and detection efficiency influence factors of the system were also analyzed. The results show that the target site mutation model detection shows good repeatability and high resolution by using this system, the sequence with only one base mutation can also be resolved very well. Mainly factors affecting the detection efficiency is amplification fragment length and mutations location in the amplified fragments. This study also using SSCP genotyping method to detect rice endogenous gene modification sites. The research selected three rice endogenous gene modification sites OsDEP1, OsYSA2 and OsROC5 to application of SSCP genotyping method. The protoplast samples and stable transformation of mutant plant samples of these 3 sites are assayed by SSCP genotyping assay respectively. The results show that the SSCP genotyping method can only genotyping of OsDEP1 site which with higher conversion efficiency in protoplast samples. For OsDEP1, OsYSA2 and OsROC5 mutant individuals not only have achieved efficient genotype, but also distinguished the different types of gene targeted mutagenesis.