Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Schematic illustration of the design and workflow. ( A ) Engineering guide RNA to make it responsive to small molecules. The gRNA scaffold contains multiple well-defined structural motifs, including a repeat:anti-repeat duplex and three stem–loops. NCD and its hydrogen-bonding pattern to the introduced G-G mismatch sites are demonstrated. The structural unit of N-acyl-2-amino-1,8-naphthyridine contains three hydrogen-bonding groups that are fully complementary to that of G. ( B ) Illustration of interference of Cas9-mediated DNA cleavage by RNA-binding small molecules. The Cas9 complex with designer gRNAs still retains wild-type levels of activity, while the exposure of this complex to MBLs leads to evident inhibition of DNA cutting activities. The protospacer adjacent motif (PAM) is indicated. Red rectangles: 2-amino-1,8-naphthyridine.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: RNA Binding Assay, Activity Assay, Inhibition

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Rational gRNA engineering for switching CRISPR/Cas9. ( A ) Sequence and structure analysis of gRNA scaffold (tracrRNA demonstration). We have identified a total of three motifs with close resemblance with the units possessing affinity for MBL binding. ( B ) MBLs are used to introduce structural constraints into gRNAs through hydrogen bonding and stacking. Sequence modification sites are indicated in red. Red rectangles: 2-amino-1,8-naphthyridine.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: CRISPR, Sequencing, Binding Assay, Introduce, Modification

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of designer sgRNAs for switching CRISPR/Cas9 Reactions were performed as described in the Experimental Section. Uncleaved SLX4IP DNA (773 bp) cut to shorter cleavage fragments (441 bp and 332 bp) are demonstrated. All samples were tested in three biological replicates. Image of representative data is shown here. ( A ) Sanger sequencing analysis of selected sgRNAs. The sites for sequence modification are indicated. ( B ) The tolerance of Cas9 to each designer sgRNA. Lane 1: target control; lane 2: Cas9-only control; lane 3 contains original sg- SLX4IP ; lanes 4–10 contain designer sgRNAs harboring different MBL-binding units; lane 11: DNA marker (GeneRuler 100-bp DNA Ladder). ( C ) Responsiveness of designer sgRNAs to different MBLs. Lane 1: no Cas9 control; lanes 2–5 contain original sg- SLX4IP ; lanes 6–10, 11–15 contain sg- SLX4IP -S2c; lane 16: DNA marker. ( D ) The NCD-dependent inhibition of CRISPR/Cas9 with single-site variants. Lane 1: no Cas9 control; lanes 2–3 contain original sg- SLX4IP ; lanes 4–9 contain sg- SLX4IP -S1a; lanes 10–15 contain sg- SLX4IP -S1b; lanes 16–21 contain sg- SLX4IP -S1c; lane 22: DNA marker. ( E ) The NCD-dependent inhibition of CRISPR/Cas9 with multi-nucleotide variants. Lane 1: no Cas9 control; lanes 2–3 contain original sg- SLX4IP ; lanes 4–8 contain sg- SLX4IP -S2a; lanes 9–13 contain sg- SLX4IP -S2b; lanes 14–18 contain sg- SLX4IP -S2c; lanes 19–23 contain sg- SLX4IP -S3; lane 24: DNA marker.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: CRISPR, Sequencing, Modification, Binding Assay, Marker, Inhibition

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of designer tracrRNAs for switching CRISPR/Cas9 Reactions were performed as described in the Experimental Section. All samples were tested in three biological replicates. Image of representative data was shown here. ( A ) Illustration of MBL-responsive CRISPR/Cas9 with designer tracrRNAs. Red rectangles: 2-amino-1,8-naphthyridine. ( B ) The tolerance of Cas9 to each designer tracrRNA. Lane 1: target control; lane 2: Cas9-only control; lane 3 contains cr- SLX4IP and original tracrRNA; lanes 4–10 contain cr- SLX4IP and designer tracrRNAs harboring different MBL-binding units; lane 11: DNA marker (GeneRuler 100-bp DNA Ladder). ( C ) Dose-dependent response of the indicated tracrRNAs to each MBL. Lane 1: no Cas9 control; lanes 2–5 contain cr- SLX4IP and original tracrRNA; lanes 6–10, 11–15 contain cr- SLX4IP and tracrRNA-S2c; lane 16: DNA marker. ( D ) Effects of NCD on the function of tracrRNA and its single-site variants. Lane 1: no Cas9 control; lanes 2–3 contain cr- SLX4IP and original tracrRNA; lanes 4–9 contain cr- SLX4IP and tracrRNA-S1a; lanes 10–15 contain cr- SLX4IP and tracrRNA-S1b; lanes 16–21 contain cr- SLX4IP and tracrRNA-S1c; lane 22: DNA marker. ( E ) Effects of NCD on the function of tracrRNA and its multi-nucleotide variants. Lane 1: no Cas9 control; lanes 2–3 contain cr- SLX4IP and original tracrRNA; lanes 4–8 contain cr- SLX4IP and tracrRNA-S2a; lanes 9–13 contain cr- SLX4IP and tracrRNA-S2b; lanes 14–18 contain cr- SLX4IP and tracrRNA-S2c; lanes 19–23 contain cr- SLX4IP and tracrRNA-S3; lane 24: DNA marker.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: CRISPR, Binding Assay, Marker

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of designer sgRNAs in a stable Cas9-expressing cell line Cellular studies were performed using HeLa-OC cells as described in the Experimental Section. The treatment for each sample is indicated by the signs at the bottom of each lane. All samples were tested in three biological replicates. Image of representative data is shown here. ( A ) Editing of SLX4IP gene in HeLa-OC cells using the indicated sgRNAs. Lane 1: target control; lane 2: no sgRNA control; lane 3 contains original sg- SLX4IP ; lanes 4–10 contain designer sgRNAs harboring different MBL-binding units; lane 11: DNA marker (GeneRuler 100-bp DNA Ladder). ( B ) The effect of sequence modification on the function of sgRNAs in cells. ( C ) Ligand control of designer sgRNAs in HeLa-OC cells. Hela-OC cells were exposed to the NCD ligand for 24 h before being harvested for DNA cleaving activity assessments. Lane 1: target control; lanes 2–3: no sgRNA control; lanes 4–5 contain original sg- SLX4IP ; lanes 6–9 contain sg- SLX4IP -S1b; lanes 10–13 contain sg- SLX4IP -S1c; lanes 14–17 contain sg- SLX4IP -S2c; lane 18: DNA marker. ( D ) Bar graph shows the effect of NCD on the function of sgRNAs in HeLa-OC cells. In each group, the indel formation of NCD-treated cells were compared to that of mock-treated cells. P values less than 0.05 are given one asterisk, and P values <0.001 are given three asterisks. For (A) and (C), uncleaved SLX4IP DNA (773 bp) cut to shorter cleavage fragments (441 bp and 332 bp) are demonstrated. For (B) and (D), data represent the mean of three replicates and were shown as mean ± SEM.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: Expressing, Binding Assay, Marker, Sequencing, Modification, Activity Assay

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of plasmid-based gene editing in human cells Cellular studies were performed as described in the Experimental Section. The plasmids and/or sgRNAs were delivered into HeLa cells before the treatment with NCD. Hela cells were exposed to the NCD ligand for 24 h before being harvested for DNA cleaving activity assessments. All samples were tested in three biological replicates. Image of representative data is shown here. ( A ) Ligand control of the hybrid system with IVT sgRNAs and Cas9-only plasmids. Lane 1: target control; lanes 2–3: no sgRNA control; lanes 4–5 contain PX165 and sg- SLX4IP ; lanes 6–9 contain PX165 and sg- SLX4IP -S1b; lanes 10–13 contain PX165 and sg- SLX4IP -S1c; lanes 14–17 contain PX165 and sg- SLX4IP -S2c; lane 18: DNA marker (GeneRuler 100-bp DNA Ladder). ( B ) Bar graph shows the effect of NCD on the function of the hybrid system (IVT sgRNAs and PX165). ( C ) Ligand control of all-in-one plasmids with MBL-binding units. Lane 1: target control; lanes 2–3: no plasmid control; lanes 4–5 contain PX459- SLX4IP ; lanes 6–9 contain PX459-S2c- SLX4IP ; lane 10: DNA marker. ( D ) Bar graph shows the effect of NCD on the function of all-in-one plasmids. For (A) and (C), uncleaved SLX4IP DNA (773 bp) cut to shorter cleavage fragments (441 bp and 332 bp) are demonstrated. For (B) and (D), the data are presented as the means ± SEM from three independent experiments. In each group, the indel formation of NCD-treated cells were compared to that of mock-treated cells. P values less than 0.05 are given one asterisk, P values <0.01 are given two asterisks, and P values <0.001 are given three asterisks.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: Plasmid Preparation, Activity Assay, Marker, Binding Assay

Journal: Infectious Disease Clinics of North America

Article Title: USE OF THE CLINICAL MICROBIOLOGY LABORATORY FOR THE DIAGNOSIS AND MANAGEMENT OF INFECTIOUS DISEASES RELATED TO THE ORAL CAVITY

doi: 10.1016/S0891-5520(05)70108-2

Figure Lengend Snippet: LABORATORY DETECTION OF ORAL MICROBIAL PATHOGENS

Article Snippet: In a national survey, the College of American Pathologists (CAP) sent 2692 laboratories a swab seeded with S. pyogenes to perform a rapid detection test with the kit used at their institution.

Techniques: Infection, Virus, Diagnostic Assay

Journal: Nature Communications

Article Title: Glycosylation of serine/threonine-rich intrinsically disordered regions of membrane-associated proteins in streptococci

doi: 10.1038/s41467-025-58692-8

Figure Lengend Snippet: a – c Representative TLC analysis of [ 3 H]Glc-lipids extracted from in vitro incubations of UDP-[ 3 H]Glc with the membrane fractions isolated from S pyogenes WT ( a ), Δ gtrB ( b ), and Δ gtrB :p gtrB ( c ). d Coomassie-stained gel of S. pyogenes membrane proteins purified by conA affinity chromatography. Proteins in excised bands were identified by LC-MS/MS analysis as described in Methods. The major identified proteins are indicated. e , f Immunoblot analysis of PrsA1 ( e ) and PknB ( f ) in S. pyogenes WT, Δ gtrB , Δ gtrB :p gtrB , and Δ gtrB :p sccN using specific antibodies. Anti-PrsA1 antibodies also recognize PrsA2. Proteins were separated on a 4-12% SurePAGE™– Bis-Tris gel (Genscript) in MES buffer in d – f . The experiments were performed independently three times in a , b , c , e , and f , and two times in d , yielding the same results. A representative image from one experiment is shown. g Topology of extracytoplasmic domains of S. pyogenes conA-bound proteins. * The cytoplasmic domain of PknB is omitted for clarity. Just one monomer of the dimer for PrsA1 and PknB is shown for clarity. PrsA1 is depicted as a diacylated lipoprotein because lipoproteins in streptococci are diacylated as N-acyl-glyceryl-cysteine , . Source data for a – f are provided as a Source Data file. Schematic in g was created in BioRender .

Article Snippet: Rabbit polyclonal antibodies against the S. pyogenes PrsA1 (1:1000) and PrsA2 (1:1000) homologs were provided by Dr. Yung-Chi Chang .

Techniques: In Vitro, Membrane, Isolation, Staining, Purification, Affinity Chromatography, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

doi: 10.1073/pnas.0804754105

Figure Lengend Snippet: High-throughput screen for chemical inhibitors that displace a Pol III peptide from the β-clamp. (A) Titration of E. coli β into TAMN-labeled Pol III C-terminal 20-mer peptide is monitored by fluorescence anisotropy. (B) Inhibition of DNA replication by compounds identified in the peptide-displacement assay. The plot shows the percentage of inhibition of E. coli DNA Pol I Klenow versus β-dependent synthesis by Pol III* in the presence of 20 μM compound. (C) Chemicals (i.e., at 50 μM) that displace E. coli Pol III α-peptide from E. coli β were tested for ability to displace S. pyogenes Pol C peptide from S. pyogenes β.

Article Snippet: HPLC-purified peptides were from Bio-Synthesis Inc.: Pol III C-terminal 9-mer (double-underlined), 20-mer (underlined), 30-mer (italic) peptides ( GATWRVSPSD RLLNDLRGLIG SEQVELEFD ), Pol III internal β-binding motif (IGQADMFGV), Pol II C-terminal 10-mer (TAMN-T 1 L 2 M 3 T 4 G 5 Q 6 L 7 G 8 L 9 F 10 ), S. pyogenes PolC C-terminal 20-mer (TAMN-MGILGNMPEDNQLSLFDDFF), and S. aureus PolC 20-mer (TAMN-DELGSLPNLPDKAQLSIFDM).

Techniques: High Throughput Screening Assay, Titration, Labeling, Fluorescence, Inhibition