Journal: bioRxiv

Article Title: Anionic polymers stabilize Cas9 ribonucleoprotein nanoparticles to improve direct protein delivery and genome editing efficiencies in plant protoplasts

doi: 10.1101/2025.02.02.635105

Figure Lengend Snippet: Gene editing efficiencies of PEG-transfected Cas9 RNPs to protoplasts that were (a) formulated with or without the anionic polymer PGA to N. benthamiana protoplasts. For all editing results, editing efficiencies were quantified using CRISPResso2 software to analyze NGS results. Mean and SD of three technical replicates are shown. Unpaired t test was used to calculate P value of indicated comparisons. ** P < 0.01. (b) Viability of A. thaliana protoplasts pre- and post-transfection was evaluated using FDA staining. Data is shown for three individual wells for each condition, representing biological replicates of each treatment. The mean and SD of FDA positive cells are plotted for each well, with each data point representing the percentage of FDA positive cells in a single field of view in each well. Representative images are shown in SI Fig. 3. (c) Gene editing efficiencies of PEG-transfected Cas9 RNPs that were formulated with anionic polymer PGA alone, A5K alone, or both PGA and A5K to A. thaliana protoplasts. (d) The hydrodynamic radius of RNP-nanoparticles in NEBuffer3 was evaluated by DLS. (e) Cas9 RNP editing efficiencies in N. benthamiana protoplasts with peptides conjugated or unconjugated (free). Mean and SD of three technical replicates are shown.

Article Snippet: Stocks of sgRNA and apo-Cas9 were thawed on ice, diluted individually to 15μM and 10μM in NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 1 mM DTT, pH = 7.9 at 25°C) respectively, and brought to room temperature.

Techniques: Transfection, Polymer, Software, Staining

Journal: bioRxiv

Article Title: Anionic polymers stabilize Cas9 ribonucleoprotein nanoparticles to improve direct protein delivery and genome editing efficiencies in plant protoplasts

doi: 10.1101/2025.02.02.635105

Figure Lengend Snippet: Gene editing efficiencies of PEG-transfected Cas9 RNPs to protoplasts that were (a) formulated with or without the anionic polymer PGA to N. benthamiana protoplasts. For all editing results, editing efficiencies were quantified using CRISPResso2 software to analyze NGS results. Mean and SD of three technical replicates are shown. Unpaired t test was used to calculate P value of indicated comparisons. ** P < 0.01. (b) Viability of A. thaliana protoplasts pre- and post-transfection was evaluated using FDA staining. Data is shown for three individual wells for each condition, representing biological replicates of each treatment. The mean and SD of FDA positive cells are plotted for each well, with each data point representing the percentage of FDA positive cells in a single field of view in each well. Representative images are shown in SI Fig. 3. (c) Gene editing efficiencies of PEG-transfected Cas9 RNPs that were formulated with anionic polymer PGA alone, A5K alone, or both PGA and A5K to A. thaliana protoplasts. (d) The hydrodynamic radius of RNP-nanoparticles in NEBuffer3 was evaluated by DLS. (e) Cas9 RNP editing efficiencies in N. benthamiana protoplasts with peptides conjugated or unconjugated (free). Mean and SD of three technical replicates are shown.

Article Snippet: In brief, fresh aliquots of 40 μM apo-Cas9 were buffer-exchanged to remove TCEP using Zebra Spin Desalting Columns with a 7K MWCO (ThermoFisher Scientific).

Techniques: Transfection, Polymer, Software, Staining

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: Schematic representation of the CRISPR-mediated immunity acquired in bacteria through (1) adaptation, integration of foreign DNA as a spacer (S0) in CRISPR loci after leader sequence (L) flanked by direct repeats (R); (2) expression, decoding of CRISPR array into pre-crRNA, Cas protein(s), and tracrRNA followed by subsequent processing of pre-crRNA into crRNA; and (3) interference, Cas9 in complex with crRNA:tracrRNA neutralizes the reinfection. The Cas9:crRNA:tracrRNA complex recognizes the foreign DNA by using PAM sequence and DNA:RNA complementarity and triggers Cas9-catalyzed DNA cleavage (the cleavage site is indicated by black arrows), thus providing immunity against viral reinfection. The target DNA (tDNA) strand is shown in orange, and the nontarget strand is shown in yellow. The PAM sequence is shown in the nontarget DNA (ntDNA) strand. The adaptation, expression, and interference stages are represented with gray, green, and blue arrows, respectively.

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques: CRISPR, Bacteria, Sequencing, Expressing

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: Simple overview of CRISPR/Cas classification. Cas proteins (oval) involved in various stages of CRISPR function are represented in different colors: gray (adaptation), green (expression), and blue (interference). The Cas9 protein in the type-II system is the focus of this Review, highlighted with a dark border.

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques: CRISPR, Expressing

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: (a) Different domains of Cas9 protein and their lengths are represented with different colors. (b) X-ray structure of Cas9 endonuclease adopted from PDB 5F9R (resolution = 3.4 Å), captured in a precleavage state containing sgRNA (orange) and intact dsDNA (yellow). The structure on the right side is rotated by 180° around the axis of sgRNA. The protein is demonstrated as a transparent surface, while the dsDNA and sgRNA are visualized as cartoons.

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques:

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: Comparison of the X-ray crystal structures of Cas9 in different states: (a) apo Cas9 (PDB ID 4CMQ ), (b) the Cas9:sgRNA complex (PDB ID 4ZT0 ), (c) the Cas9:sgRNA:tDNA complex with the PAM sequence of the nontarget strand (PDB ID 4UN3 ), and (d) the Cas9:sgRNA:dsDNA complex (PDB ID 5F9R ). The domains showing the major conformational changes between two consecutive states are highlighted as opaque cartoons (also indicated by colored arrows), while the domains having similar structures between two consecutive stages of Cas9 are represented as transparent cartoons. The target and nontarget strands of DNA are denoted as tDNA and ntDNA, respectively.

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques: Comparison, Sequencing

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: Schematic representation of the structure of the CRISPR/Cas9 R-loop complex and the key interactions in the Cas9:crRNA:tracrRNA:dsDNA complex. The tDNA and ntDNA are represented in yellow and orange, respectively. The spacer, repeats, and tracrRNA regions of sgRNA are represented in red, pink, and yellow, respectively. Key interactions associated with nucleotide binding and catalysis are visualized as stick representations in the circles. Mg 2+ ions were placed in the RuvC and HNH catalytic sites on the basis of models predicted by Zuo and Liu, 2016, and Zhu et al., 2019, respectively.

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques: CRISPR, Binding Assay

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: (a) Schematic representation of the effect of base-pairing mismatches on HNH activation and off-target effects. (b) Outline of the effect of reducing nonspecific interactions in altering ntDNA flexibility and Cas9 specificity. (c) Protein mutations that have been reported to alter Cas9 specificity. Asterisks (*) denote mutations that reduce the off-target effect, and the blue “▲” denotes a mutation that increases Cas9’s specificity toward the PAM (NGG) motif.

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques: Activation Assay, Mutagenesis

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: Pictorial representation highlighting the important findings from the Molecular Dynamics studies. (a) Targeted Molecular Dynamics (tMD) of Cas9:sgRNA → apoCas9 and Cas8:sgRNA:dsDNA → Cas9:sgRNA:tDNA:incomplete-ntDNA. The arrows depict the conformational changes of REC I–III and the rotation of the HNH domains. (b) Distance between catalytic H840 residue and tDNA cleavage site (scissile phosphate) in the presence and absence of complete ntDNA. (c) Roles of different REC domains of Cas9 in nucleotide recognition and catalysis. (d) PAM facilitates allosteric signaling (K775-Q771 and R905-E584 interactions) and establishes the correlation between the HNH and RuvC domains involving the L1 and L2 loops.

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques: Residue

Journal: ACS Omega

Article Title: Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

doi: 10.1021/acsomega.2c05583

Figure Lengend Snippet: Progress of Molecular Dynamics (MD) Simulation-Based Studies on the CRISPR/Cas9 System, the MD System Used, the Methodology Adopted, Their Inferences Obtained, and the Supporting Experimental Studies

Article Snippet: 2017 , Apo Cas9 (PDB 4CMQ ), Cas9:RNA (PDB 4ZT0 ), Cas9:DNA (PDB 4UN3 ), Cas9:precat (PDB 5F9R ) , Gaussian-accelerated Molecular Dynamics (GaMD), targeted Molecular Dynamics (TMD) , •upon transition from apo-Cas9 to RNA-bound Cas9, the recognition lobe adopts a more open conformation forming a positively charged cavity for RNA binding , •FRET , .

Techniques: CRISPR, Sequencing, Binding Assay, Cleavage Assay, Derivative Assay, Activation Assay, RNA Binding Assay, Structural Proteomics, Mutagenesis, Cryo-EM Sample Prep, Modification, Isolation, In Vitro, Activity Assay, Variant Assay, Protein-Protein interactions, Depletion Assay, DNA Sequencing