GSE119994
Targeted sequencing by a CRISPR-assistant target enrichment-sequencing (CATE-seq)
Targeted sequencing of genome is essential to the basic biological research and biomedicine. Therefore, various targeted enrichment methods have been developed for this end, in which various hybridization-based methods have been most widely used. However, the current hybridization-based methods (both on solid or in solution) are still restricted by the intrinsic shortcomings of nucleic acid hybridization. In this study, by combining an engineered dCas9-sgRNA with widely used magnetic isolation, we developed a new strategy to enrich target genomic DNAs in high efficiency. In this strategy, target DNA was firstly specifically recognized and bound by a complex of dCas9 and capture sgRNA (csgRNA). The DNA-dCas9-csgRNA complex was then captured on magnetic beads through the annealing of elongated 3' end of csgRNA with single-stranded capture oligonucleotides coupled on streptavidin-coated magnetic beads. We thus named the strategy as CRISPR-assistant target enrichment (CATE). The enriched DNAs can be analyzed by the next generation sequencing (NGS). We thus named the technique as CATE-seq. Used the technique, we successfully enriched 35 target exons of 6 genes in 6 cell lines by using 54 csgRNA. We found that the target genomic DNA fragments such as exons could be efficiently enriched and analyzed by CATE-seq. The technique has several significant advantages over the current hybridization-based methods, including high simplicity, specificity, sensitivity, and throughput. This study therefore provides a new powerful tool for targeted sequencing. Overall design: we developed a new dCas9/sgRNA-based target enrichment technique by combining dCas9-sgRNA with magnetic separation. The 3' end of sgRNA was redesigned to add a short capture sequence in complementary with a single-stranded capture oligonucleotide coupled on magnetic beads. The special sgRNA was named as capture sgRNA (csgRNA). The single-stranded capture oligonucleotides is anchored on the surface of magnetic beads by biotin-streptavidin interaction, in which the single-stranded capture oligonucleotide was modified by biotin and streptavidin was coated on the surface of magnetic beads. In order to enrich target DNA fragments from an input DNA library, the input DNA library was firstly incubated with the dCas9-csgRNA to form DNA-dCas9-csgRNA complex, which were them captured by the capture oligonucleotides-coupled magnetic beads via annealing of capture sequence of csgRNA with capture oligonucleotides. The captured DNA-dCas9-csgRNA complex can be easily isolated from input DNA library by a magnetic separation. The enriched DNA can be analyzed by next-generation sequencing. We named this technique as CATE-seq. We used this technique successfully enriched 35 exons of 6 genes in 6 cell lines, indicating the feasibility, reliability, specificity, and simplicity of the new target enrichment sequencing technique.