Novel Strategy On Target Enrichment Based On Sequence-specific DNA Binding Protein

Target enrichment is widely used in medical diagnostics and biology,especially in the field of Next-Generation Sequencing(NGS).Compared to research on specific genomic sites,whole genomic sequencing is a time-consuming and expensive procedure,and Next-Generation Sequencing(NGS)are increasingly dependent on the ability of the specific data captured from complex samples quickly and accurately.Target sequencing of specific genomic sites could increase the sequencing depth with limited sequencing data,and could greatly decrease the consumption of time and cost.Based on CRISPR/dCas9 system this research develop a simple and rapid,efficient and specific method of targeted enrichment,named CRISPR assisted target enrichment(CATE).To validate the method,28 captured sgRNA(csgRNA)were designed for capture the exons of ATK1,BRAC1,TP53 gene.With the dCas9/csgRNA complex,the target exons were recognized and identified from the Tn5 transposon cleaved 293 T cell genomic DNA,then purified using the biotin-streptavidin system.With the clone-sequencing,80% of the captured sequence were located in the target exon regions.In order to research the potential applications of CATE,367 captured sgRNAs were designed to capute exons from 186 genes.And enriching experiment was performed with 14 cell-free DNA(cfDNA)samples from esophageal cancer patient and healthy persons,and sequenced with NGS.By comparing the sequencing data of before and after the capture,it was found that,in a relatively complex environment,CATE can increase the ratio of target DNA,from 0.12% in origin sample up to 94.47% in enriched sample.These results indicate that the CATE could capture the target DNA in cfDNA effectively.Subsequently,in order to reveal the defference of mutations in cfDNA between the healthy persons and esophageal cancer patients.2,302 csgRNAs for exons of 450 gene were designed,and were used to capture the target in 15 cell-free DNA(cfDNA)samples which from esophageal cancer patients and healthy persons.The results showed that CATE could increase the ratio of target DNA,from 0.05% in origin sample up to 97.89% in enriched sample,and 39 mutations that potentially relevant with esophageal cancer were identified.This study developed a novel CRISPR-based capture method for targeted capture sequencing.Totally 29 cfDNA samples were analyzed,the results showed that the method could capture the target sequence accurately and also could be used to identify the mutations in the cfDNA of esophageal cancer patients and healthy persons,which was with great protential in testing the esophageal cancer.The method provide a new strategy for enriching target sequencing and mutation identification.