29. Nanomapping with Cas9: A new high-resolution and efficient method for characterizing chromosomal rearrangements

      Improvements in the genomic tools available for detecting chromosomal rearrangements (CREA) have resulted in the recognition of many new pathognomonic CREA/cancer associations, with these discoveries providing a basis for the development of new treatment modalities and prognostic stratifications. Excitement regarding the application of this CREA knowledge for patient management is accompanied by the recognition of a growing need for an improved assay that can yield a precise, high-resolution characterization of these CREA in a manner that is time-, cost- and specimen-use-efficient. To address this need, we developed “nanomapping”: a technique in which we (1) develop a unique “barcode” for a region of interest using nuclease-inhibited Cas9 molecules as stable and specific programmable labels; and (2) measure the lengths of DNA fragments and distances between Cas9 landmarks with nanoscale accuracy using high-speed atomic force microscopy. By comparing the barcode to a reference sequence, one can identify alterations in “long read” (≥100kb) DNA molecules at high speed and low cost ($1 per sample). To adapt this technique for CREA characterization, we: (1) used inverse PCR to amplify a translocation site of interest with multiplexed endonucleases to yield a characteristic profile of amplicon lengths; (2) nanomapped the resulting amplicons; and (3) compared the nanomaps to predicted reference maps generated in silico to obtain precise (high accuracy/specificity) identification of the chromosomal origin/breakpoints of fusion partners. As “proof of principle” for the utility of this new assay, to date, we have correctly identified IGH fusion partners in 4 neoplasms (including IGH/BCL2 translocations in 3/3 specimens and an IGH/MAFB translocation in 1/1 specimen). In summary, our data demonstrates the feasibility of using nanomapping to characterize CREA that have recurrent breakpoints. Moreover, it not only provides a means for enhancing our efficiency/accuracy in known CREA identification, but has potential to allow for the recognition of novel CREA.
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