DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle

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.

Fig. 1

High-speed AFM measurement of Cas9-labeled DNA. a Cas9 labeling chemistry and sample preparation work flow: (I) incubate target DNA with Cas9–sgRNA and fix with formaldehyde, (II) remove unbound Cas9–sgRNA complexes and deposit molecules on mica, and (III) image with HS-AFM and measure locations of labels on DNA. b High-speed contact mode AFM, with a coffee mug for scale. The zoom-in view of the x–y flexure scanner shows the relative positions of the sample, cantilever, and laser vibrometer objective. c As configured, the HS-AFM produces two 16-bit, 1000 × 1000 pixel images per second

Fig. 2

sgRNA–Cas9 complexes on DNA. a Map and the corresponding HS-AFM image of Cas9-labeled BRCA1 amplicon. Red tick marks on the map indicate locations of the Alu repeat-targeted sgRNA-binding sites. In the image, sgRNA–Cas9 protein complexes appear as 3-nm-tall bumps on the DNA backbone. The amplicon sequence includes five perfect matches to the 20-bp sgRNA sequence and five single base-pair mismatches. Single base-pair mismatches label at roughly one-half the frequency of perfect matches. The star (*) indicates Cas9 bound at locations with a single mismatch, while all other locations are perfect matches. b, c Cas-9-labeled TERT and HER2 amplicons. The sharp bend induced in the DNA backbone by the sgRNA–Cas9 complex is evident in both examples

Fig. 3

HS-AFM accurately localizes sgRNA–Cas9 complexes on DNA. a Measured vs. expected position of the Cas9 labels determined for BRCA1, TERT-HER2, and ladder constructs. Error bars represent the 95% confidence interval of the mean. For the TERT-HER2 and ladder data points, the symbol diameter is larger than the 95% confidence interval span. The expected size and sample standard deviation (σ) are indicated at three points for reference. b Sizing closely spaced ladder constructs, end-labeled with Cas9. Vertical red lines inside symbols represent the 95% confidence interval of the mean. See Methods for measurement detail

Fig. 4

Mapping BCL2–IGH translocations with Cas9. a Maps of PCR amplicons from wild-type BCL2 and IGH with Cas9 labels (different colors for emphasis). The two labels on BCL2 straddle the 300-bp MBR translocation hotspot. b HSAFM images of labeled amplicons. c HSAFM-determined maps of the translocation breakpoints from a follicular lymphoma cell line DOHH2, and the seven patient samples. Identity of the Cas9 labels is determined by measuring their position from the end of the molecule. Maps were confirmed with 10 or more molecules each. In all seven patent samples, the translocation breakpoint mapped to the major breakpoint region (MBR) of BCL2 and the J_H region of IGH

Fig. 5

Detecting Cas9 labels with a DVD player part used as a displacement sensor. a Photo of the DVD player optical pickup unit showing the location of the laser-focusing objective and the signal and control connection point. Behind the objective lens is the 650-nm-wavelength diode laser, quadrant photodiode, and associated optics. b Schematic of the HS-AFM scan stage with the DVD optical pickup used as the cantilever displacement sensor. c An example image of TERT amplicons labeled with two Cas9 proteins. This image was obtained by averaging five full frames collected at the maximum frame rate of 2 frames per second. d Typical height cross section from double-labeled molecules obtained with the DVD optical pickup. e Histograms of Cas9 marker spacing measured for populations of TERT and HER2 molecules. The white asterisk indicates the expected value