CRISPR-Cas9 Targeted Enrichment and Next-Generation Sequencing for Mutation Detection.

Despite the rapid application of next-generation sequencing (NGS) technologies, target sequencing in regions of the genome is often required to diagnose many genetic diseases. Target enrichment can be an effective factor in reducing the cost of sequencing and the duration of sequencing. Recently, several clustered system regularly interspaced short palindromic repeats (CRISPR)-based methods (amplification-free sequencing) have been developed to target enrichment in combination with one of the NGS platforms. CRISPR-based target enrichment strategies act as an auxiliary tool to improve NGS analytical performance, thereby indirectly facilitating nucleic acid detection. The direct DNA cleavage approach by CRISPR-CRISPR-associated (Cas) at genome-specific sites enhances the possibility of separating native large fragments from disease-related genomic regions. The CRISPR-Cas can isolate the target region without any amplification; subsequently, long-read sequencing technologies were also implemented. These methods, as promising tools, have the ability to assess genetic and epigenetic composition for clinical application and treatment responses in cancer precision medicine. By modifying CRISPR-based enrichment protocols, it is possible to identify different types of mutations, including structural variants, short tandem repeats, fusion genes, and mobile elements. The Cas9 can specifically eliminate wild-type sequences, and it also enables the enrichment and detection of small amounts of tumor DNA fragments among the highly heterogeneous fragments of wild-type DNA.