Tonight I watched the Oxford Nanopore Technologies Nanopore Learning Knowledge Exchange session on Cas9 PCR-free Enrichment. I think this kit has been discontinued, yet it is important to learn how it works. Anne-Marie Wadsworth presented the method of PCR-free enrichment using Cas9. Wadsworth noted that ONT has developed a method to use Cas9 to target a region of interest (ROI), attach sequencing chemistry, and add to a nanopore device for sequencing. The PCR-free system allows for detection of highly repetitive reads, long gene targets that are difficult to PCR and modified bases or methylation patterns. Wadsworth explained that the CRISPR RNAs (crRNAs) program Cas9 to bind and cleave DNA sites that are identical or highly similar to the crRNA sequence. Cas9 is loaded with crRNA and in combination with the trans-activating CRISPR RNA tracrRNA, it forms a complex. The protospacer-adjacent motif (PAM sequence NGG) is recognized and cleavage occurs at a distance. The crRNA probe is the same as the target sequence. Wadsworth recommended a minimum of 1 kb flanking region between the ROI and probe target sequence. Wadsworth emphasized the importance of the crRNA design. ONT recommends an “excision approach” for regions <20 kb. Both ends of the target must be known. Two cuts are made on each side for redundancy. When the region is less than 20 kb, a probe design with a single cut and read out is recommended. Tiling approaches with multiple probes can be used with regions >20 kb. To assist with designing the crRNA sequences, the CHOPCHOP web-based design tool is recommended. CHOPCHOP has settings for Nanopore sequencing and will identify PAM and off target sequences. There is also an IDT ordering tool. An ONT probe design tool was mentioned by Wadsworth. The first step of the protocol is to dephosphorylate 5′ to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles bind and cleave the region of interest. The ligatable 5′ phosphate is used to prepare blunt ends for sequencing adapter. Wadsworth noted that the throughput may seem lower but the region of interest is enriched in coverage. Pore occupancy, noted Wadsworth, is driven by the ends with adapter. Wadsworth and ONT recommend using metrics to measure the enrichment of the ROI. The percentage of reads on target should be increased. Wadsworth shared several examples and data. Wadsworth also encouraged the use of spike in controls. The sample preparation is under two hours! I wonder if they will have a kit or option for V14 chemistry!
