Nina Gill
Research Mentor: Brian Athey
Mentor Department: Computational Medicine & Bioinformatics, Medicine
Author(s): Nina Gill, Rachel Case, Greg Farnum, Yisang Moon, Isaac Farnum, Zachary Freeman, Monica Holmes, Brian Athey
Session: Session 2 (10:00 AM – 10:50 AM)
Presentation Type: Poster 82
Abstract
Single-molecule sequencing enables the direct analysis of individual DNA molecules and is performed using technologies such as Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio). These platforms sequence individual DNA molecules continuously, generating long reads that enable high-resolution analysis of genomic DNA. However, many applications require interrogation of specific genomic regions rather than whole-genome coverage. Targeted sequencing allows specific loci to be selectively analyzed. One of the most effective strategies for targeted sequencing involves the nuclease Cas9. Cas9 is traditionally characterized as a blunt-end endonuclease, producing double-stranded DNA breaks without generating nucleotide overhangs. For optimal ONT and PacBio library preparation, predictable blunt cleavage is critical, as sequencing adaptors require proper ligation to DNA ends. However, sequencing results generated using PacBio revealed uneven coverage between targeted genomic regions, with one region exhibiting approximately 60× coverage and another only 30× coverage. This discrepancy, along with emerging reports in the literature, suggests that Cas9 cleavage may not always produce blunt ends. To better understand Cas9 in vitro cutting activity, this study examined Cas9 cutting efficiency and evaluated whether Cas9 produces staggered DNA ends through two main experiments. In the cutting efficiency experiment, Cas9 activity was measured across 15 single guide RNAs (sgRNAs) targeting different locations within the gene CYP2D6. Cutting efficiency varied between sgRNAs, ranging from 64.4% to 100%. To determine the presence of overhangs, DNA fragments labeled with distinct fluorophores on each strand were cleaved with Cas9. Capillary electrophoresis was then used to analyze the resulting fragments, allowing detection of differences in fragment length that would indicate staggered rather than blunt DNA ends. A more detailed understanding of Cas9 end structure has important implications for improving targeted sequencing approaches and optimizing adaptor ligation strategies for ONT and PacBio sequencing.
