DNA Interrogation Research Articles

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA engineering begins with local helix distortion by transient CRISPR-Cas protein binding.

Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

Convergent evolution of BRCA2 reversion mutations under therapeutic pressure by PARP inhibition and platinum chemotherapy

Reversion mutations that restore wild-type function of the BRCA gene have been described as a key mechanism of resistance to Poly(ADP-ribose) polymerase (PARP) inhibitor therapy in BRCA-associated cancers. Here, we report a case of a patient with metastatic castration-resistant prostate cancer (mCRPC) with a germline BRCA2 mutation who developed acquired resistance to PARP inhibition. Extensive genomic interrogation of cell-free DNA (cfDNA) and tissue at baseline, post-progression, and postmortem revealed ten unique BRCA2 reversion mutations across ten sites. While several of the reversion mutations were private to a specific site, nine out of ten tumors contained at least one mutation, suggesting a powerful clonal selection for reversion mutations in the presence of therapeutic pressure by PARP inhibition. Variable cfDNA shed was seen across tumor sites, emphasizing a potential shortcoming of cfDNA monitoring for PARPi resistance. This report provides a genomic portrait of the temporal and spatial heterogeneity of prostate cancer under the selective pressure of a PARP inhibition and exposes limitations in the current strategies for detection of reversion mutations.

RET-Altered Cancers-A Tumor-Agnostic Review of Biology, Diagnosis and Targeted Therapy Activity.

RET alterations, such as fusions or mutations, drive the growth of multiple tumor types. These alterations are found in canonical (lung and thyroid) and non-canonical (e.g., gastrointestinal, breast, gynecological, genitourinary, histiocytic) cancers. RET alterations are best identified via comprehensive next-generation sequencing, preferably with DNA and RNA interrogation for fusions. Targeted therapies for RET-dependent cancers have evolved from older multikinase inhibitors to selective inhibitors of RET such as selpercatinib and pralsetinib. Prospective basket trials and retrospective reports have demonstrated the activity of these drugs in a wide variety of RET-altered cancers, notably those with RET fusions. This paved the way for the first tumor-agnostic selective RET inhibitor US FDA approval in 2022. Acquired resistance to RET kinase inhibitors can take the form of acquired resistance mutations (e.g., RET G810X) or bypass alterations.

Structural basis for Cas9-mediated DNA interrogation and editing with 5′ truncated sgRNAs

Structural basis for Cas9-mediated DNA interrogation and editing with 5′ truncated sgRNAs

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

Utilization of the Tapestri Platform for Quantification of Single Cell Vector Copy Number in CAR T Cell Products

Chimeric antigen receptor (CAR) T cell immunotherapies have been transformative solutions to treat both adult and pediatric patients with a variety of cancers. Given that CAR T cell therapies involve genetic alterations of a patient's T cells by introduction of the CAR into host cells using lentiviral vectors, the quality of CAR T cell therapies are extensively regulated to warrant safety. Key safety and efficacy attributes need to be accurately measured prior to reintroducing modified T cells back into patients, including CAR transgene copy number or viral vector copy number (VCN). We have previously compared digital PCR (dPCR) strategies to the historical standard release assay of quantitative PCR (qPCR) for VCN quantification given that dPCR is known to provide absolute quantification of target nucleic acid sequences with many advantages over qPCR in regards to simplicity, reproducibility, lower limit of detection, and definitive quantification. We were able to demonstrate that dPCR produces accurate, precise, and reproducible CAR T cell VCN measurements when compared to qPCR. However, these conventional PCR methods measuring vector copy number are either population averaged or involve labor-intensive and time-consuming single cell colony picking and outgrowth, with respective drawbacks in resolution and sample representation. Here, we introduce the Tapestri platform, to measure thousands of single cell level VCN with accuracy and precision. The Tapestri platform workflow is enabled by its two droplet steps. First, thousands of cells are each microfluidically encapsulated in a first droplet where lysis of the cells occur, releasing DNA from its heterochromatin state for uniform DNA interrogation. Then, the individual lysates are uniquely barcoded and target sequences amplified via multiplexed PCR inside each droplet. The final products are sequenced on an Illumina sequencing instrument. Single cell VCN Tapestri data was generated and orthogonally compared with dPCR and matches with the bulk measurement on clonal Jurkat cell lines (VCN=2) mixed with control cell lines (VCN=0) at a range of different ratios with accuracy and precision. We demonstrate that the Tapestri platform provides absolute quantification of CAR transgenes at the single cell level and has the potential to provide more precise measurements of CAR T cell therapeutic product attributes prior to release to patients. Future studies will evaluate the utility of this Tapestri platform in measuring both CAR transgene copies and surface expression/density of the CAR at the single cell level, further improving upon current CAR T cell product release assays.

Association of fetal fraction with hypertensive disorders of pregnancy incidence and disease severity

Hypertensive disorders of pregnancy contribute to maternal and offspring morbidity and mortality. Studies suggest that a lower early pregnancy fetal fraction is associated with an increased risk of hypertensive disorders of pregnancy. However, maternal obesity significantly affects fetal fraction and is a risk factor for hypertensive disorders of pregnancy. We determined the association between fetal fraction (using a standardized single-institution platform, including male and female fetuses) and hypertensive disorders of pregnancy, stratified by obesity status. Second, we evaluated differences in total cell-free DNA concentration and correlation of fetal fraction with clinical markers of hypertensive disorders of pregnancy severity. This was a retrospective, single-institution study of a previously validated cell-free DNA-based noninvasive prenatal screening assay of 1058 samples. Maternal body mass index at the time of noninvasive prenatal screening was assessed, and hypertensive disorders of pregnancy were confirmed by a detailed medical record review. Differences in fetal fraction and total cell-free DNA concentration between the groups were assessed with univariate analyses. Multivariable regression was used to evaluate the association between fetal fraction and hypertensive disorders of pregnancy, adjusted for body mass index, maternal age, gestational age at noninvasive prenatal screening, and fetal sex. The association between fetal fraction and hypertensive disorders of pregnancy among individuals with obesity (body mass index, ≥30 kg/m2) and individuals without obesity (body mass index, <30 kg/m2) was investigated while controlling for the aforementioned covariates. Lastly, multivariable linear regression was used to evaluate the association between fetal fraction and clinical markers of hypertensive disorders of pregnancy severity. We identified individuals with (n=117) and without (n=941) hypertensive disorders of pregnancy with noninvasive prenatal screening drawn before 20 weeks of gestation and with fetal fraction and body mass index data available. Those with hypertensive disorders of pregnancy had a lower fetal fraction (10.2%±4.2% vs 11.6%±4.7%; P<.01), without differences in total cell-free DNA concentration (P=.14). When groups were stratified by obesity status, this relationship was only valid for individuals without obesity (P=.02). Only when logistic regression analysis was restricted to individuals without obesity did the likelihood of hypertensive disorders of pregnancy rise with decreasing fetal fraction (odds ratio, 0.93; 95% confidence interval, 0.88-0.99; P=.02). In addition, fetal fraction was inversely associated with maximum systolic blood pressure at the time of hypertensive disorders of pregnancy only in the population without obesity (β, -0.08; 95% confidence interval, -0.147 to -0.01; P=.02). Although a lower fetal fraction is associated with the development of hypertensive disorders of pregnancy, the use of this parameter for the prediction may be problematic in individuals with obesity, as obesity has such a profound effect on fetal fraction. However, we uniquely noted that among individuals without obesity, fetal fraction is lower for those that develop hypertensive disorders of pregnancy and lower fetal fraction increases the odds of hypertensive disorders of pregnancy development. Lastly, low fetal fraction in the population without obesity that developed hypertensive disorders of pregnancy was associated with higher systolic blood pressure at the time of hypertensive disorders of pregnancy, an important clinical marker of hypertensive disorders of pregnancy severity. As analytical approaches of cell-free DNA interrogation advance, the prediction of placental-mediated disorders with first-trimester sampling is likely to improve, although this may remain challenging in gravidas with obesity, a cohort at high risk of developing hypertensive disorders of pregnancy.

Single-molecule analysis of DNA interrogation by Cas9 and Cas12a on supercoiled DNA

Cas9 and Cas12a are CRISPR endonucleases that interrogate DNA sequences by forming an R-loop structure containing an RNA:DNA heteroduplex before cleaving a target site that matches a guide RNA. The sequence specificity of R-loop formation and cleavage has enabled wide applications in genetic engineering. R-loop substeps are affected by mismatches with the guide RNA and by target DNA topology, which must be understood to arrive at mechanochemical descriptions of the mechanisms underlying specificity in different CRISPR endonucleases.

Efficient DNA interrogation of SpCas9 governed by its electrostatic interaction with DNA beyond the PAM and protospacer

Streptococcus pyogenes Cas9 (SpCas9), a programmable RNA-guided DNA endonuclease, has been widely repurposed for biological and medical applications. Critical interactions between SpCas9 and DNA confer the high specificity of the enzyme in genome engineering. Here, we unveil that an essential SpCas9–DNA interaction located beyond the protospacer adjacent motif (PAM) is realized through electrostatic forces between four positively charged lysines among SpCas9 residues 1151–1156 and the negatively charged DNA backbone. Modulating this interaction by substituting lysines with amino acids that have distinct charges revealed a strong dependence of DNA target binding and cleavage activities of SpCas9 on the charge. Moreover, the SpCas9 mutants show markedly distinguishable DNA interaction sites beyond the PAM compared with wild-type SpCas9. Functionally, this interaction governs DNA sampling and participates in protospacer DNA unwinding during DNA interrogation. Overall, a mechanistic and functional understanding of this vital interaction explains how SpCas9 carries out efficient DNA interrogation.

Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling

The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.

CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions with DNA.

CRISPR-Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR-Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR-Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. The DNA interrogation and R-loop formation involve several distinct steps the molecular details of which are not fully understood. This chapter describes a recently developed fluorometric Cas beacon assay that may be used for measuring of specific affinity of various CRISPR-Cas complexes for unlabeled target DNA and model DNA probes. The Cas beacon approach also can provide a sensitive method for monitoring the kinetics of assembly of CRISPR-Cas complexes.

ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions.

Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a)

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Duplex DNA Destabilization by Type V CRISPR-Cas Nucleases during Interrogation of DNA

The prokaryotic CRISPR-Cas effector complexes locate their target sites by scanning and interrogating the genomic DNA. The type II and type V CRISPR-Cas nucleases cleave double-stranded DNA bearing sequences complementary to guide segments of CRISPR RNAs (crRNA) flanked by conserved PAM sequences; they have been successfully used for genome editing in various organisms. Unpairing of a short PAM-proximal segment of DNA protospacer is a critical event that may determine the overall rate of target location. We sought to elucidate mechanistic details of initial DNA interrogation steps by type V CRISPR-Cas nucleases Cas12a and Cas12b (also known as Cpf1 and C2c1, respectively). Using fluorometric and biochemical assays, we studied interactions of Cas12a and Cas12b effectors with a set of model DNA substrates that mimic likely early intermediates on the pathway to the final R-loop complexes. Consistent with reported data on the mechanism of DNA interrogation by type II Cas9 nuclease, we find that binding of type V effectors to PAM favors separation of several adjacent to PAM DNA protospacer base pairs even in the absence of pairing between the crRNA guide and the protospacer. The separation is mediated in part by effector affinity for non-target DNA strand nucleotides in a single-stranded conformation. In contrast, effector interactions with the nearest to PAM target DNA strand nucleotides are energetically unfavorable in the type V early intermediate complexes, presumably due to a steric clash between the protein and DNA backbones. We suggest that these unfavorable interactions destabilize the DNA duplex and thus facilitate the DNA interrogation process. We discuss the similarities and differences between the mechanisms of DNA interrogation by Cas9, Cas12a and Cas12b effectors.

Mechanism of Genome Interrogation: How CRISPR RNA-Guided Cas9 Proteins Locate Specific Targets on DNA

The ability to precisely edit and modify a genome opens endless opportunities to investigate fundamental properties of living systems as well as to advance various medical techniques and bioengineering applications. This possibility is now close to reality due to a recent discovery of the adaptive bacterial immune system, which is based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that utilize RNA to find and cut the double-stranded DNA molecules at specific locations. Here we develop a quantitative theoretical approach to analyze the mechanism of target search on DNA by CRISPR RNA-guided Cas9 proteins, which is followed by a selective cleavage of nucleic acids. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the target search is presented. It is found that the location of specific sites on DNA by CRISPR Cas9 proteins is governed by binding first to protospacer adjacent motif sequences on DNA, which is followed by reversible transitions into DNA interrogation states. In addition, the search dynamics is strongly influenced by the off-target cutting. Our theoretical calculations allow us to explain the experimental observations and to give experimentally testable predictions. Thus, the presented theoretical model clarifies some molecular aspects of the genome interrogation by CRISPR RNA-guided Cas9 proteins.

Abstract 2441: NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein

Abstract Introduction: Development of improved cancer diagnostics and therapeutics requires detailed understanding of the genomic, transcriptomic, and proteomic profiles in the tumor microenvironment. Current technologies can excel at measuring a single analyte, but it remains challenging to simultaneously collect high-throughput DNA, RNA, and protein data from small samples. We have developed an approach that uses optical barcodes to simultaneously profile DNA, RNA, and protein from as little as 5ng DNA, 25ng RNA, and 250ng protein or just 2 5µm FFPE slides, and simplifies data analysis by generating digital counts for each analyte. Methods: The approach uses paired capture and reporter oligonucleotide probes and optical barcodes to enumerate up to 800 targets. The platform was initially developed to measure RNA, and we have adapted it to measure DNA single nucleotide variants (SNVs), proteins, and phospho-proteins. SNVs are detected by direct hybridization of sequence discriminating probes to the wild-type and mutant sequence of interest. Proteins are detected via binding of oligonucleotide-conjugated antibodies. Results: Combinations of DNA, RNA, and protein in biological and experimental contexts. SNV probes are able to detect variant alleles down to 5% abundance within a wild type population and can discriminate variants within mutation hotspots. It was &amp;gt;96% accurate at identifying variants from samples displaying a range of allele frequencies and DNA integrity when benchmarked against next-generation sequencing. Protein detection has been developed for cell surface, cytosolic, and nuclear proteins, as well as phospho-proteins. It was validated against flow cytometry, western blot, and mass spectrometry using cell lines with ectopic target expression and primary cells. To demonstrate concurrent measurement of DNA, RNA, and protein from a single system, BRAFWT or BRAFV600E cell lines were treated with the BRAFV600E inhibitor vemurafenib and the MEK inhibitor trametinib. We measured the allele usage at the BRAFV600 locus, as well as BRAFV600E dependent changes in mRNA expression, protein expression and protein phosphorylation in a single experiment. Conclusions: 3D Biology has several advantages over other analytical approaches. Direct, single-molecule digital counting allows detection over a broad dynamic range with high reproducibility, often over 98% concordance between technical replicates. The simultaneous interrogation of DNA, RNA, and protein maximizes the amount of data obtained from precious samples and minimizes instrumentation demands by leveraging a single detection platform. The 3D Biology approach allows holistic, digital analysis of biological samples with high specificity and precision. This technology is currently available for research use, but may also have clinical application in the future. Citation Format: Chris Lausted, Yong Zhou, Jinho Lee, Christopher Vellano, Karina A. Eterovic, Ping Song, Lin-ya Tang, Gloria Fawcett, Tae-Beom Kim, Ken Chen, Gary Geiss, Gavin Meredith, Qian Mei, Gokhan Demirkan, Dwayne Dunaway, Dae Kim, P. Martin Ross, Elizabeth Manrao, Nathan Elliott, Sarah Warren, Christina Bailey, Chung-Ying Huang, Joseph Beechem, Gordon Mills, Leroy Hood. NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2017-2441

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.