Elucidation of the molecular mechanism of the breakage-fusion-bridge (BFB) cycle using a CRISPR-dCas9 cellular model.

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanisms for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrated that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we showed that targeting dCas9 to telomeres using sgTelo impeded DNA replication at telomeres and induced a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigated the genome-wide features of telomeres in the dCas9/sgTelo cells and observed a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-.Consistently, we also observed an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncovered many novel molecular and structural features of the ITCB and demonstrated that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.

Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM) Can Potentially Define the ALT Positivity of Cancer.

Telomeres play an essential role in protecting the ends of linear chromosomes and maintaining the integrity of the human genome. One of the key hallmarks of cancers is their replicative immortality. As many as 85-90% of cancers activate the expression of telomerase (TEL+) as the telomere maintenance mechanism (TMM), and 10-15% of cancers utilize the homology-dependent repair (HDR)-based Alternative Lengthening of Telomere (ALT+) pathway. Here, we performed statistical analysis of our previously reported telomere profiling results from Single Molecule Telomere Assay via Optical Mapping (SMTA-OM), which is capable of quantifying individual telomeres from single molecules across all chromosomes. By comparing the telomeric features from SMTA-OM in TEL+ and ALT+ cancer cells, we demonstrated that ALT+ cancer cells display certain unique telomeric profiles, including increased fusions/internal telomere-like sequence (ITS+), fusions/internal telomere-like sequence loss (ITS-), telomere-free ends (TFE), super-long telomeres, and telomere length heterogeneity, compared to TEL+ cancer cells. Therefore, we propose that ALT+ cancer cells can be differentiated from TEL+ cancer cells using the SMTA-OM readouts as biomarkers. In addition, we observed variations in SMTA-OM readouts between different ALT+ cell lines that may potentially be used as biomarkers for discerning subtypes of ALT+ cancer and monitoring the response to cancer therapy.

Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.

Single-molecule analysis of subtelomeres and telomeres in Alternative Lengthening of Telomeres (ALT) cells.

BACKGROUND: Telomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887-898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. RESULTS: Our results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats. CONCLUSION: Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.

A micropatterned substrate for on-surface enzymatic labelling of linearized long DNA molecules.

Optical mapping of linearized DNA molecules is a promising new technology for sequence assembly and scaffolding, large structural variant detection, and diagnostics. This is currently achieved either using nanochannel confinement or by stretching single DNA molecules on a solid surface. While the first method necessitates DNA labelling before linearization, the latter allows for modification post-linearization, thereby affording increased process flexibility. Each method is constrained by various physical and chemical limitations. One of the most common techniques for linearization of DNA uses a hydrophobic surface and a receding meniscus, termed molecular combing. Here, we report the development of a microfabricated surface that can not only comb the DNA molecules efficiently but also provides for sequence-specific enzymatic fluorescent DNA labelling. By modifying a glass surface with two contrasting functionalities, such that DNA binds selectively to one of the two regions, we can control DNA extension, which is known to be critical for sequence-recognition by an enzyme. Moreover, the surface modification provides enzymatic access to the DNA backbone, as well as minimizing non-specific fluorescent dye adsorption. These enhancements make the designed surface suitable for large-scale and high-resolution single DNA molecule studies.