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DNA Unwinding by CRISPR-Cas9 Studied using Site-Directed Spin Labeling
238a
Monday, February 29, 2016
activity in the nucleus of live cells, and open the path to study connections between nuclear organization and metabolism. Grants: NIH- DA036408, AG041504, P41-GM103540 and P50-GM076516 1181-Pos Board B158 Examining Tale Protein Binding Kinetics and Site Competition using Single Molecule Imaging Max Kushner1, Alexander Van Slyke1, Fabio Rinaldi2, Avtar Singh3, John Lis1, Adam Bogdanove2, Warren Zipfel3. 1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA, 2Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY, USA, 3Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA. Transcription activator-like effector (TALE) proteins are a set of regulatory proteins that can be programmed to bind specifically any DNA sequence of choice. Despite the obvious potential of TALE proteins as fluorescence imaging and site specific genome editing tools, very little is known about their binding kinetics and affinity in the presence of other DNA-binding proteins that may be competing for the same or nearby sites. Utilizing a polyethylene glycol (PEG) coated flow cell we are able to observe TALE protein binding to a tethered target DNA. We compare the binding kinetics both on and off target to study TALE protein binding specificity. To investigate the usefulness of TALE proteins for live cell imaging of targeted loci we examined TALE binding kinetics in the presence of competition and interactions with DNA binding proteins. 1182-Pos Board B159 A Molecule-Scale View of the Structure and Specificity of the RNA-Guided Endonuclease Cas9 Eric A. Josephs1, D. Dewran Kocak2, Christopher J. Fitzgibbon1, Joshua McMenemy2, Charles A. Gersbach3, Piotr E. Marszalek1. 1 Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA, 2Biomedical Engineering, Duke University, Durham, NC, USA, 3 Biomedical Engineering; Center for Genomic and Computational Biology, Duke University, Durham, NC, USA. CRISPR-associated endonuclease Cas9 binds to and cleaves DNA at sites dictated by a variable sequence in an RNA molecule that is bound to it. This ability of Cas9 to be directed by a modular ‘single-guide’ RNA molecule (sgRNA) to target nearly any DNA sequence has recently been exploited for a number of emerging biological and biomedical applications: in particular, Cas9-based technologies have been re-appropriated in heterologous systems for remarkably facile genomic engineering. Therefore, understanding the molecular-scale factors that drive Cas9 to target these sites and the nature of its reported off-target activity are of paramount importance for its practical usage and will be vital in developing new ways to improve its specificity. Using atomic force microscopy (AFM), we are able to directly resolve individual Cas9 and nuclease-inactivate dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind to targeted or off-target sequences, which we are able to modulate performance and specificity by re-engineering sgRNAs with variant structures, and map the structural properties of Cas9 and dCas9 to their respective binding sites. In combination with kinetic Monte Carlo (KMC) simulations, these results reveal a progressive conformational transformation at DNA sites with increasing sequence similarity to its target is identified and provide evidence of a conformational gating’ governing Cas9 and dCas9 specificity. Additionally, we are able to correlate the results of our KMC simulations and model for Cas9-DNA interactions with reported off-target cleavage rates of Cas9 in vivo. Our results reveal new methodologies to engineer guide RNA sequences and structures that improve Cas9 and dCas9 specificity. 1183-Pos Board B160 DNA Unwinding by CRISPR-Cas9 Studied using Site-Directed Spin Labeling Narin S. Tangprasertchai, Carolina Vazquez Reyes, Xiaojun Zhang, Peter Z. Qin. Chemistry, University of Southern California, Los Angeles, CA, USA. In a type II clustered, regularly interspersed, short palindromic repeats (CRISPR) system, RNAs derived from the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs sequence-specifically. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms; however, its mechanism of function at the molecular level remains to be fully understood. A key step in Cas9
target selection is the unwinding of the target DNA duplex to allow formation of a three-stranded R-loop, in which the guide segment of the RNA is base-paired to the protospacer segment residing at one of the DNA strands. Here, we investigate DNA deformation and unwinding in Streptococcus pyogenes Cas9 (SpyCas9) complexes using the method of site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy. Nucleotide-independent nitroxide labels were attached at selected sites of a target DNA duplex, and labeled DNAs were assembled with SpyCas9 and the corresponding RNA, resulting in minimal perturbation of native activity. EPR measurements revealed distinct increases in inter-strand distances along the target DNA duplex in the Cas9 complex, consistent with DNA deformation and unwinding. Importantly, the inter-strand distances increased non-uniformly along the protospacer segment of the DNA, indicating a varying degree of DNA unwinding within the region complementary to the guide RNA. Additional work is ongoing to more clearly understand the mechanism of DNA unwinding and its connection with target cleavage by Cas9. 1184-Pos Board B161 Investigation of the Cas9 Mediated DNA Cleavage using Time-Lapse AFM Imaging Suleyman Ucuncuoglu1,2, Ozgur Sahin1,2. 1 Biological Sciences, Columbia University, New York, NY, USA, 2Physics, Columbia University, New York, NY, USA. Bacteria and archaea have a defense mechanism for the viral DNA invasion that is facilitated by the clustered regularly interspaced short palindromic repeats (CRISPR), and the CRISPR associated (Cas) proteins. The viral infections trigger their adaptive immune system by insertion the foreign DNA segments into the CRISPR loci followed by transcribing the small RNAs with Cas proteins. In particular, type II system cas protein (cas9) associates with the guide RNA to target and silence the viral infections by cleaving the both strands of DNA. The ability of substituting the viral specific RNA with another desired guide RNA makes cas9:RNA system an interest of not only bacterial immunity, but also genetic engineering. Recently, several reports demonstrated the successful repression of genes by means of cas9 enzyme when the target sequence is closer to the promoter site. Despite rapidly increasing number of studies related to the cas9 application, kinetics of cas9 based cleavage mechanism is weakly understood. Using Atomic Force Microscopy (AFM), we monitored the cas9 cleavage activity of the target DNA upon introducing the cas9:RNA complexes to the aqueous environment. We will present our kinetic analysis of cas9 nuclease activity. Our approach may shed light on the possible single-turnover kinetic feature of the cas9 enzyme and help improve cas9-mediated gene editing and therapeutic developments. 1185-Pos Board B162 DNA Binding Fluorescent Proteins for the Direct Visualization of Large DNA Molecules Seonghyun Lee, Kyubong Jo. Sogang Univ, Seoul, Korea, Republic of. Fluorescent proteins that also bind DNA molecules are promising reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘‘paints’’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for partial intercalating between DNA bases (1). Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd =14.7 mM, 7-8 bp/ FP-DBP of base occupancies per dye) to confluently stain DNA molecules and such binding is reversible via pH shifts of buffer solution. These proteins offer robust advantages for lack of fluorophore mediated photocleavage, which makes them ideal staining reagents for imaging of DNA molecules over extended time periods even without anti-bleaching agent because the DNA binding moiety of the FP is separated from the fluorophore moiety. Further, the staining with FP-DBPs does not perturb polymer contour lengths, showing that the l DNA monomer is about 48 502 bp 0.34 nm/bp = 16.5 mm long, while most of the intercalating dyes, such as EtBr and YOYO-1, are known to distort the DNA structure and increase its full contour length. Moreover, the most significant advantage of FP-DBPs for DNA staining is its application to live cells, or entire living organisms. In a unique fashion, the FP-DBP fluorescence localized in three or four discrete regions within the bacterial cells: one, or two spots in the middle, and some at both ends of cells, as well as DAPI stained bacterial cells.
Monday, February 29, 2016
activity in the nucleus of live cells, and open the path to study connections between nuclear organization and metabolism. Grants: NIH- DA036408, AG041504, P41-GM103540 and P50-GM076516 1181-Pos Board B158 Examining Tale Protein Binding Kinetics and Site Competition using Single Molecule Imaging Max Kushner1, Alexander Van Slyke1, Fabio Rinaldi2, Avtar Singh3, John Lis1, Adam Bogdanove2, Warren Zipfel3. 1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA, 2Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY, USA, 3Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA. Transcription activator-like effector (TALE) proteins are a set of regulatory proteins that can be programmed to bind specifically any DNA sequence of choice. Despite the obvious potential of TALE proteins as fluorescence imaging and site specific genome editing tools, very little is known about their binding kinetics and affinity in the presence of other DNA-binding proteins that may be competing for the same or nearby sites. Utilizing a polyethylene glycol (PEG) coated flow cell we are able to observe TALE protein binding to a tethered target DNA. We compare the binding kinetics both on and off target to study TALE protein binding specificity. To investigate the usefulness of TALE proteins for live cell imaging of targeted loci we examined TALE binding kinetics in the presence of competition and interactions with DNA binding proteins. 1182-Pos Board B159 A Molecule-Scale View of the Structure and Specificity of the RNA-Guided Endonuclease Cas9 Eric A. Josephs1, D. Dewran Kocak2, Christopher J. Fitzgibbon1, Joshua McMenemy2, Charles A. Gersbach3, Piotr E. Marszalek1. 1 Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA, 2Biomedical Engineering, Duke University, Durham, NC, USA, 3 Biomedical Engineering; Center for Genomic and Computational Biology, Duke University, Durham, NC, USA. CRISPR-associated endonuclease Cas9 binds to and cleaves DNA at sites dictated by a variable sequence in an RNA molecule that is bound to it. This ability of Cas9 to be directed by a modular ‘single-guide’ RNA molecule (sgRNA) to target nearly any DNA sequence has recently been exploited for a number of emerging biological and biomedical applications: in particular, Cas9-based technologies have been re-appropriated in heterologous systems for remarkably facile genomic engineering. Therefore, understanding the molecular-scale factors that drive Cas9 to target these sites and the nature of its reported off-target activity are of paramount importance for its practical usage and will be vital in developing new ways to improve its specificity. Using atomic force microscopy (AFM), we are able to directly resolve individual Cas9 and nuclease-inactivate dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind to targeted or off-target sequences, which we are able to modulate performance and specificity by re-engineering sgRNAs with variant structures, and map the structural properties of Cas9 and dCas9 to their respective binding sites. In combination with kinetic Monte Carlo (KMC) simulations, these results reveal a progressive conformational transformation at DNA sites with increasing sequence similarity to its target is identified and provide evidence of a conformational gating’ governing Cas9 and dCas9 specificity. Additionally, we are able to correlate the results of our KMC simulations and model for Cas9-DNA interactions with reported off-target cleavage rates of Cas9 in vivo. Our results reveal new methodologies to engineer guide RNA sequences and structures that improve Cas9 and dCas9 specificity. 1183-Pos Board B160 DNA Unwinding by CRISPR-Cas9 Studied using Site-Directed Spin Labeling Narin S. Tangprasertchai, Carolina Vazquez Reyes, Xiaojun Zhang, Peter Z. Qin. Chemistry, University of Southern California, Los Angeles, CA, USA. In a type II clustered, regularly interspersed, short palindromic repeats (CRISPR) system, RNAs derived from the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs sequence-specifically. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms; however, its mechanism of function at the molecular level remains to be fully understood. A key step in Cas9
target selection is the unwinding of the target DNA duplex to allow formation of a three-stranded R-loop, in which the guide segment of the RNA is base-paired to the protospacer segment residing at one of the DNA strands. Here, we investigate DNA deformation and unwinding in Streptococcus pyogenes Cas9 (SpyCas9) complexes using the method of site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy. Nucleotide-independent nitroxide labels were attached at selected sites of a target DNA duplex, and labeled DNAs were assembled with SpyCas9 and the corresponding RNA, resulting in minimal perturbation of native activity. EPR measurements revealed distinct increases in inter-strand distances along the target DNA duplex in the Cas9 complex, consistent with DNA deformation and unwinding. Importantly, the inter-strand distances increased non-uniformly along the protospacer segment of the DNA, indicating a varying degree of DNA unwinding within the region complementary to the guide RNA. Additional work is ongoing to more clearly understand the mechanism of DNA unwinding and its connection with target cleavage by Cas9. 1184-Pos Board B161 Investigation of the Cas9 Mediated DNA Cleavage using Time-Lapse AFM Imaging Suleyman Ucuncuoglu1,2, Ozgur Sahin1,2. 1 Biological Sciences, Columbia University, New York, NY, USA, 2Physics, Columbia University, New York, NY, USA. Bacteria and archaea have a defense mechanism for the viral DNA invasion that is facilitated by the clustered regularly interspaced short palindromic repeats (CRISPR), and the CRISPR associated (Cas) proteins. The viral infections trigger their adaptive immune system by insertion the foreign DNA segments into the CRISPR loci followed by transcribing the small RNAs with Cas proteins. In particular, type II system cas protein (cas9) associates with the guide RNA to target and silence the viral infections by cleaving the both strands of DNA. The ability of substituting the viral specific RNA with another desired guide RNA makes cas9:RNA system an interest of not only bacterial immunity, but also genetic engineering. Recently, several reports demonstrated the successful repression of genes by means of cas9 enzyme when the target sequence is closer to the promoter site. Despite rapidly increasing number of studies related to the cas9 application, kinetics of cas9 based cleavage mechanism is weakly understood. Using Atomic Force Microscopy (AFM), we monitored the cas9 cleavage activity of the target DNA upon introducing the cas9:RNA complexes to the aqueous environment. We will present our kinetic analysis of cas9 nuclease activity. Our approach may shed light on the possible single-turnover kinetic feature of the cas9 enzyme and help improve cas9-mediated gene editing and therapeutic developments. 1185-Pos Board B162 DNA Binding Fluorescent Proteins for the Direct Visualization of Large DNA Molecules Seonghyun Lee, Kyubong Jo. Sogang Univ, Seoul, Korea, Republic of. Fluorescent proteins that also bind DNA molecules are promising reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘‘paints’’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for partial intercalating between DNA bases (1). Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd =14.7 mM, 7-8 bp/ FP-DBP of base occupancies per dye) to confluently stain DNA molecules and such binding is reversible via pH shifts of buffer solution. These proteins offer robust advantages for lack of fluorophore mediated photocleavage, which makes them ideal staining reagents for imaging of DNA molecules over extended time periods even without anti-bleaching agent because the DNA binding moiety of the FP is separated from the fluorophore moiety. Further, the staining with FP-DBPs does not perturb polymer contour lengths, showing that the l DNA monomer is about 48 502 bp 0.34 nm/bp = 16.5 mm long, while most of the intercalating dyes, such as EtBr and YOYO-1, are known to distort the DNA structure and increase its full contour length. Moreover, the most significant advantage of FP-DBPs for DNA staining is its application to live cells, or entire living organisms. In a unique fashion, the FP-DBP fluorescence localized in three or four discrete regions within the bacterial cells: one, or two spots in the middle, and some at both ends of cells, as well as DAPI stained bacterial cells.