DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle

Andrey Mikheikin,Loren Picco,Anita Olsen,Andrew Yacoot,Amir Toor,James K Gimzewski,Oliver Payton,Alden Chesney,Kevin Leslie,Jason Reed,Freddie Russell-Pavier,Bud Mishra

doi:10.1038/s41467-017-01891-9

Abstract

Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM. As a proof of principle, we used this new “nanomapping” method to detect and map precisely BCL2–IGH translocations present in lymph node biopsies of follicular lymphoma patents. This HS-AFM “nanomapping” technique can be complementary to both sequencing and other physical mapping approaches.

Highlights

Progress in whole-genome sequencing using short-read (e.g.,
Despite being invented in the mid-1990s, single-molecule optical mapping has had only a limited impact on the field of human genomics compared to the more recently invented nextgeneration sequencing, primarily because it lacks sufficient resolution to deal with structurally complex regions
It is for this reason that the most successful applications of that technique in human genomics have been as a supplement to sequencing approaches (e.g., Illumina, PacBio, etc.)[4] It is possible that in time, “super-optical-resolution” techniques (e.g., PALM, STORM, etc.) will transform optical mapping

Summary

Results

We demonstrated the efficiency and precision of Cas[9] labeling using several gene-specific sgRNAs targeted to BRCA1, HER2, and TERT gene sequences (see Supplementary Fig. 1 for maps). To label the BRCA1 amplicon, we employed a single sgRNA that matches a subset of naturally occurring Alu repeats in the genomic locus (see Supplementary Fig. 2 for further exemplary images). One sgRNA species exhibited an unusually low initial labeling rate (35%); this was improved to 87% by increasing the sgRNA concentration to threefold vs standard conditions This result suggests that many DNA sequences containing a GG motif can be targeted with proper reaction optimization. Using the Cas[9] labels, we mapped a known BCL2–IGH translocation present in the follicular lymphoma cell line DOHH-2, obtained commercially, and detected similar translocations in seven lymph node tissue samples from follicular lymphoma patients (Fig. 4c). By increasing the scan speed and image size, these higher pixel rates can be translated into measurement throughput directly

Discussion

Objective lens Sample

Methods