A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation
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{"title"=>"A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation", "type"=>"journal", "authors"=>[{"first_name"=>"Iman", "last_name"=>"Farasat", "scopus_author_id"=>"25652924100"}, {"first_name"=>"Howard M.", "last_name"=>"Salis", "scopus_author_id"=>"9839371000"}], "year"=>2016, "source"=>"PLoS Computational Biology", "identifiers"=>{"scopus"=>"2-s2.0-84956760340", "sgr"=>"84956760340", "issn"=>"15537358", "doi"=>"10.1371/journal.pcbi.1004724", "pmid"=>"26824432", "isbn"=>"1553-7358 (Electronic)\\r1553-734X (Linking)", "pui"=>"608034576"}, "id"=>"b9009738-9242-3e25-ab8f-0e5953156fe2", "abstract"=>"The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9's off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits.", "link"=>"http://www.mendeley.com/research/biophysical-model-crisprcas9-activity-rational-design-genome-editing-gene-regulation-1", "reader_count"=>176, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Professor > Associate Professor"=>4, "Researcher"=>50, "Student > Doctoral Student"=>7, "Student > Ph. D. Student"=>57, "Student > Postgraduate"=>4, "Other"=>8, "Student > Master"=>25, "Student > Bachelor"=>15, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Professor > Associate Professor"=>4, "Researcher"=>50, "Student > Doctoral Student"=>7, "Student > Ph. D. Student"=>57, "Student > Postgraduate"=>4, "Other"=>8, "Student > Master"=>25, "Student > Bachelor"=>15, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>6, "Agricultural and Biological Sciences"=>72, "Chemical Engineering"=>5, "Chemistry"=>3, "Computer Science"=>6, "Engineering"=>15, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>52, "Materials Science"=>1, "Medicine and Dentistry"=>1, "Neuroscience"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Physics and Astronomy"=>10, "Social Sciences"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Social Sciences"=>{"Social Sciences"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>10}, "Unspecified"=>{"Unspecified"=>6}, "Environmental Science"=>{"Environmental Science"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>5}, "Engineering"=>{"Engineering"=>15}, "Chemistry"=>{"Chemistry"=>3}, "Neuroscience"=>{"Neuroscience"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>72}, "Computer Science"=>{"Computer Science"=>6}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>52}}, "reader_count_by_country"=>{"United States"=>7, "China"=>1, "Finland"=>1, "Taiwan"=>1, "United Kingdom"=>2, "France"=>1, "Germany"=>2}, "group_count"=>12}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/4270849"], "description"=>"<p>Parameter values used in this study.</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620450, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.t002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Parameter_values_used_in_this_study_/2620450", "title"=>"Parameter values used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270786"], "description"=>"<p>(A) Each crRNA strand is expressed with rate r<sub>crRNA</sub>. The active crRNA is formed by either hybridization of an expressed tracrRNA with an expressed precrRNA or by direct expression of a single guide RNA (sgRNA). The Cas9 endonuclease is expressed with rate r<sub>Cas9</sub>. (B) Cas9 binds to the mature crRNA with a forward kinetic association constant k<sub>f</sub>. After loading the crRNA, the structure of the Cas9:crRNA undergoes an isomerization with forward kinetic constant k<sub>I</sub> to create an active complex. N<sub>crRNA</sub>, N<sub>Cas9</sub>, N<sub>intermediate</sub>, and N<sub>Cas9:crRNA</sub> are their numbers of molecules. (C) The resulting active complex performs a 3D random walk with molar flow rate r<sub>RW</sub>. The probability that it binds to a DNA site is determined by the site sequence, including the presence of a protospacer adjacent motif (PAM), the number of same-sequence DNA sites (N<sub>target, j</sub>), and their binding free energy (ΔG<sub>target, j</sub>). (D) The formation of a stable Cas9:crRNA:DNA complex occurs in several steps: Cas9:crRNA recognizes the PAM site, unwinds the DNA duplex, and sequentially replaces DNA:DNA base pairings with RNA:DNA bases pairings in an exchange reaction to form a DNA:RNA:DNA complex, called an R-loop. The DNA target site's binding free energy to Cas9:crRNA (ΔG<sub>target</sub>) sums together its PAM interaction energy (ΔG<sub>PAM</sub>), the energy needed to unwind the supercoiled DNA (ΔΔG<sub>supercoiling</sub>), and the crRNA:DNA exchange energy during R-loop formation (ΔΔG<sub>exchange</sub>). During these steps, the Cas9:crRNA:DNA complex may dissociate with first order kinetic constant k<sub>d</sub> or it may be cleave the bound DNA site with pseudo first order kinetic constant k<sub>C</sub>. (E) After cleavage, the Cas9:crRNA:DNA complex remains bound to the cleaved DNA, and is considered a no-turnover enzyme. Additional model parameters include the DNA replication rate (μ) and the degradation or dilution rates of Cas9 (δ<sub>Cas9</sub>), crRNA (δ<sub>crRNA</sub>), and Cas9:crRNA complex (δ<sub>Cas9:crRNA</sub>).</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620387, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_multi_step_mechanism_responsible_for_Cas9_mediated_DNA_site_cleavage_/2620387", "title"=>"The multi-step mechanism responsible for Cas9-mediated DNA site cleavage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270822"], "description"=>"<p>The (A) 21 position-dependent and (B) 256 sequence-dependent free energy model coefficients were determined using either (left) 3671 <i>in vitro</i> Cas9 cleavage measurements from dataset I or the (right) 5979 <i>in vivo</i> Cas9 cleavage measurements from dataset II. Coefficients were normalized to their maximum values. White boxes show unidentifiable model parameters, based on the available measurements. (C) Comparisons between apparent and model-calculated ΔΔG<sub>exchange</sub> across all single measurements. Pearson R<sup>2</sup> is 0.74 and 0.61, respectively. All points represent single measurements from Pattanayak et. al., Hsu et. al., and Mali et. al [<a href=\"http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004724#pcbi.1004724.ref033\" target=\"_blank\">33</a>,<a href=\"http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004724#pcbi.1004724.ref037\" target=\"_blank\">37</a>,<a href=\"http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004724#pcbi.1004724.ref041\" target=\"_blank\">41</a>]. (D) An example showing how the model is used to calculate ΔΔG<sub>exchange</sub> and ΔG<sub>PAM</sub> for a specific guide RNA sequence and DNA site. The energetic contributions of the three mismatches are determined by their (A) position-dependent coefficients and their (B) dinucleotide RNA:DNA identities, using the model parameterized by (left) dataset I. The (green box) PAM sequence determines ΔG<sub>PAM</sub> using <a href=\"http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004724#pcbi.1004724.t003\" target=\"_blank\">Table 3</a>.</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620420, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Parameterized_free_energy_models_show_how_mismatched_crRNA_guide_sequences_and_DNA_site_sequences_affect_Cas9_cleavage_activity_/2620420", "title"=>"Parameterized free energy models show how mismatched crRNA guide sequences and DNA site sequences affect Cas9 cleavage activity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270855"], "description"=>"<p>The energies are average values of all combinations in the first and fifth positions. (blue) The canonical PAM sites (NGGN) are bolded. N.B: no statistically significant binding. nt: nucleotide.</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620456, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.t003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Apparent_Cas9_binding_energies_to_canonical_and_non_canonical_PAM_sites_kcal_mol_/2620456", "title"=>"Apparent Cas9 binding energies to canonical and non-canonical PAM sites (kcal/mol).", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270834"], "description"=>"<p>(A) Model-calculated target binding free energies (ΔG<sub>target</sub>) are shown across genome position, plotting only one in ten positions for improved visualization. Panels represent either the (top, blue) forward strand or (bottom, red) reverse strand of the λ-phage genome. The target binding free energies are the sum of (B) the free energy change when dCas9 binds to a PAM site (ΔG<sub>PAM</sub>), (C) the free energy change during R-loop formation at PAM-proximal sites, compared to a perfectly complementary sequence (ΔΔG<sub>exchange</sub>), and the free energy change as a result of varying DNA site superhelical density (ΔΔG<sub>supercoiling</sub>). The major on-target site λ2 is denoted by stars. A major off-target site OS1 is denoted by crosses. Here, each mismatch in the crRNA and DNA site sequences contributes up to 0.78 kcal/mol to ΔΔG<sub>exchange</sub>, depending on their distance from the PAM site. The λ-phage genome is assumed to have uniform DNA superhelical density. The model-calculated binding probabilities of (d)Cas9:crRNA<sub>λ2</sub> to all possible PAM sites are shown at (D) the initial time before any Cas9 activity or (F) after a 10 minute incubation with (d)Cas9:crRNA<sub>λ2</sub>. (E) We show the model-calculated dynamics of (d)Cas9 binding occupancy at the (black line) λ2 DNA site, the (green line) major off-target site OS1, and a (inset) single off-target site with ΔG<sub>target</sub> = 0 kcal/mol.</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620435, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Calculation_of_dCas9_crRNA_sub_2_sub_binding_occupancy_across_34_363_PAM_sites_on_a_phage_genome_/2620435", "title"=>"Calculation of dCas9:crRNA<sub>λ2</sub> binding occupancy across 34,363 PAM sites on a λ-phage genome.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270804"], "description"=>"<p>Equimolar mixtures of Cas9 and crRNA (concentrations shown) were pre-incubated for 10 minutes, followed by the addition of target DNA and measuring the amount of cleaved DNA. Normalized cleaved DNA measurements (orange circles) using 25 nM negatively supercoiled plasmid DNA are compared to normalized model-calculated amounts of cleaved DNA (lines). Data points represent single measurements from Sternberg et al. [<a href=\"http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004724#pcbi.1004724.ref038\" target=\"_blank\">38</a>].</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620405, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Parameterization_of_the_model_using_i_in_vitro_i_data_/2620405", "title"=>"Parameterization of the model using <i>in vitro</i> data.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270840"], "description"=>"<p>(A) Model-calculated distributions show the numbers of human genome DNA sites that will be cleaved with varying efficiencies when using a LTR-B crRNA with either (yellow) baseline, (blue) 10-fold lower, or (green) 10-fold higher Cas9 and crRNA concentrations. (B) The expected number of off-target indel mutations when counting sites with cleavage efficiencies higher than a cut-off value. (C) The required next-generation sequencing coverage to identify the expected number of off-target indel mutations with 99% certainty. Colors same as in A. (D) The model-calculated dynamics of human genome modification under the same three scenarios, comparing (solid lines) on-target cleavage versus (dashed lines) the ratio between on-target and total off-target cleavage (specificity).</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620441, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.g006", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Model_predictions_for_human_genome_editing_/2620441", "title"=>"Model predictions for human genome editing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270729", "https://ndownloader.figshare.com/files/4270735", "https://ndownloader.figshare.com/files/4270741", "https://ndownloader.figshare.com/files/4270747", "https://ndownloader.figshare.com/files/4270753", "https://ndownloader.figshare.com/files/4270759", "https://ndownloader.figshare.com/files/4270768", "https://ndownloader.figshare.com/files/4270783"], "description"=>"<div><p>The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits.</p></div>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620351, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>["https://dx.doi.org/10.1371/journal.pcbi.1004724.s001", "https://dx.doi.org/10.1371/journal.pcbi.1004724.s002", "https://dx.doi.org/10.1371/journal.pcbi.1004724.s003", "https://dx.doi.org/10.1371/journal.pcbi.1004724.s004", "https://dx.doi.org/10.1371/journal.pcbi.1004724.s005", "https://dx.doi.org/10.1371/journal.pcbi.1004724.s006", "https://dx.doi.org/10.1371/journal.pcbi.1004724.s007", "https://dx.doi.org/10.1371/journal.pcbi.1004724.s008"], "stats"=>{"downloads"=>4, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Biophysical_Model_of_CRISPR_Cas9_Activity_for_Rational_Design_of_Genome_Editing_and_Gene_Regulation/2620351", "title"=>"A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270843"], "description"=>"<p>(A) The dynamics of Cas9-based cleavage at DNA sites with either (blue) zero, (green) one, (red) two, or (cyan) three mismatches, comparing the effects of increasing guide RNA concentration by 10-fold, increasing the genome size by 2-fold, or increasing the cellular growth rate by 2-fold. (B) A sensitivity analysis shows how changing system parameters affect a DNA site’s steady-state cleavage efficiency in growing cells. (C) The dynamics of dCas9-based transcriptional repression (promoter activity) at DNA sites with either (blue) zero, (green) one, (red) two, or (cyan) three mismatches, performing the same comparisons as in A. (D) A sensitivity analysis shows how changing system parameters affect a DNA sites’ steady-state transcriptional repression (promoter activity) in growing cells. mm, mismatch.</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620444, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.g007", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Rational_design_of_genome_editing_and_gene_regulation_/2620444", "title"=>"Rational design of genome editing and gene regulation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270816"], "description"=>"<p>(A) The addition of target DNA sites with the same sequence sequesters the Cas9:crRNA complex, and increases the transcription rate of the promoter controlling YFP expression. (B) A comparison between model-calculated transcription rates and measured YFP expression levels when either (stars) 0, (circles) 1, (diamonds) 2, (squares) 4, or (triangles) 8 additional on-target DNA sites were added. The DNA sites’ initial superhelical densities were either (left) increased by 0.0065 per occupied site or (right) kept constant. Data points and bars represent the mean and standard deviation of 2 measurements, performed in this study.</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620414, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Parameterization_of_the_model_using_i_in_vivo_i_data_/2620414", "title"=>"Parameterization of the model using <i>in vivo</i> data.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-29 01:11:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/4270846"], "description"=>"<p>A summary of all studies used to estimate the model's parameters.</p>", "links"=>[], "tags"=>["Biophysical Model", "PAM", "guide RNAs", "crRNA expression levels", "DNA supercoiling", "Type II", "cuts DNA", "Genome Editing", "Cas 9 binding", "energy models", "Cas 9", "CRISPR", "DNA sites", "Gene Regulation", "DNA superhelical densities", "engineering applications", "gene regulation", "growth conditions", "site selection", "genome", "Rational Design", "guide RNA sequences", "novel mechanism"], "article_id"=>2620447, "categories"=>["Biophysics", "Biochemistry", "Genetics", "Molecular Biology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified"], "users"=>["Iman Farasat", "Howard M. Salis"], "doi"=>"https://dx.doi.org/10.1371/journal.pcbi.1004724.t001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_summary_of_all_studies_used_to_estimate_the_model_s_parameters_/2620447", "title"=>"A summary of all studies used to estimate the model's parameters.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2016-01-29 01:11:38"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"23", "full-text"=>"26", "pdf"=>"6", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"11", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"33", "full-text"=>"39", "pdf"=>"7", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"27", "full-text"=>"34", "pdf"=>"7", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"39", "full-text"=>"37", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"50", "full-text"=>"40", "pdf"=>"10", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"18", "full-text"=>"15", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}
  • {"unique-ip"=>"20", "full-text"=>"25", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"6", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}
  • {"unique-ip"=>"27", "full-text"=>"32", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"10"}
  • {"unique-ip"=>"26", "full-text"=>"28", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"12"}
  • {"unique-ip"=>"52", "full-text"=>"63", "pdf"=>"16", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"2", "year"=>"2020", "month"=>"2"}
  • {"unique-ip"=>"60", "full-text"=>"57", "pdf"=>"16", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"12", "supp-data"=>"1", "cited-by"=>"3", "year"=>"2020", "month"=>"3"}
  • {"unique-ip"=>"75", "full-text"=>"71", "pdf"=>"13", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"12", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2020", "month"=>"4"}
  • {"unique-ip"=>"54", "full-text"=>"64", "pdf"=>"16", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"12", "cited-by"=>"0", "year"=>"2020", "month"=>"5"}
  • {"unique-ip"=>"34", "full-text"=>"33", "pdf"=>"12", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2020", "month"=>"6"}

Relative Metric

{"start_date"=>"2016-01-01T00:00:00Z", "end_date"=>"2016-12-31T00:00:00Z", "subject_areas"=>[]}
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