CRISPR-bind: a simple, custom CRISPR/dCas9-mediated labeling of genomic DNA for mapping in nanochannel arrays

Bionano genome mapping is a robust optical mapping technology used for de novo construction of whole genomes using ultra-long DNA molecules, able to efficiently interrogate genomic structural variation. It is also used for functional analysis such as epigenetic analysis and DNA replication mapping and kinetics. Genomic labeling for genome mapping is currently specified by a single strand nicking restriction enzyme followed by fluorophore incorporation by nick-translation (NLRS), or by a direct label and stain (DLS) chemistry which conjugates a fluorophore directly to an enzyme-defined recognition site. Although these methods are efficient and produce high quality whole genome mapping data, they are limited by the number of available enzymes—and thus the number of recognition sequences—to choose from. The ability to label other sequences can provide higher definition in the data and may be used for countless additional applications. Previously, custom labeling was accomplished via the nick-translation approach using CRISPR-Cas9, leveraging Cas9 mutant D10A which has one of its cleavage sites deactivated, thus effectively converting the CRISPR-Cas9 complex into a nickase with customizable target sequences. Here we have improved upon this approach by using dCas9, a nuclease-deficient double knockout Cas9 with no cutting activity, to directly label DNA with a fluorescent CRISPR-dCas9 complex (CRISPR-bind). Unlike labeling with CRISPR-Cas9 D10A nickase, in which nicking, labeling, and repair by ligation, all occur as separate steps, the new assay has the advantage of labeling DNA in one step, since the CRISPR-dCas9 complex itself is fluorescent and remains bound during imaging. CRISPR-bind can be added directly to a sample that has already been labeled using DLS or NLRS, thus overlaying additional information onto the same molecules. Using the dCas9 protein assembled with custom target crRNA and fluorescently labeled tracrRNA, we demonstrate rapid labeling of repetitive DUF1220 elements. We also combine NLRS-based whole genome mapping with CRISPR-bind labeling targeting Alu loci. This rapid, convenient, non-damaging, and cost-effective technology is a valuable tool for custom labeling of any CRISPR-Cas9 amenable target sequence.