Journal: bioRxiv

Article Title: Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

doi: 10.1101/2020.11.17.386797

Figure Lengend Snippet: A. PCR genotyping of ESC clones for insertion of the H2B or nucleolar H2B sequences into Rosa26 locus. het.: heterozygotic insertion; hom.: homozygotic insertion; WT: wild type ESCs; M: DNA marker. B. Life cell images showing GFP signal in HEK293T transfected with the hsp-TetO-GFP-Dam-DD-EF1 α -puro-T2A-TetR-KRAB plasmid and treated without or with 1 μg/ml doxycycline (Dox) and 1 μM Shield1 for 15 hours.

Article Snippet: ESCs were co-transfected with a plasmid expressing the Cas9 proteins and the sgRNA guide sequence targeting the Rosa26 locus (Genome CRISPRTM mouse ROSA26 safe harbor gene knock-in kit, SH054, GeneCopoeia) and the HDR repair template plasmid containing either H2B-Dam or H2B-Dam-NoLS constructs flanked by the homology arms with a molar ratio of 1:3.

Techniques: Clone Assay, Marker, Transfection, Plasmid Preparation

Journal: Cell Genomics

Article Title: Binding domain mutations provide insight into CTCF’s relationship with chromatin and its contribution to gene regulation

doi: 10.1016/j.xgen.2025.100813

Figure Lengend Snippet:

Article Snippet: pEN114 - pCAGGS-Tir1-V5-BpA-Frt-PGK-EM7-PuroR-bpA-Frt-Rosa26 , Nora et al. , Addgene #92143.

Techniques: Magnetic Beads, Ligation, Expressing, Mutagenesis, Recombinant, Plasmid Preparation, Software

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW , and LRRK2 KW . ( B and C ) Close-up of the inhibitor binding pocket from cryo–electron microscopy (cryo-EM) maps and models of LRRK2 RCKW bound to the type I inhibitor MLi-2 [Protein Data Bank (PDB): 8TXZ] (B) and type II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange, C-lobe; light orange, N-lobe; black, DYG motif; gray, G-loop; green, activation loop. ( D ) Scheme depicting our hybrid design strategy to develop potent type II inhibitors targeting LRRK2.

Article Snippet: The LRRK2 KW (RRID: Addgene_228879) or LRRK2 RCKW (RRID: Addgene_226783) expression constructs (table S4) encoding a Tobacco Etch Virus (TEV) cleavable N-terminal His 6 -Z-tag were expressed in SF9 insect cells (RRID:CVCL_0549) after baculoviral transfection as described previously ( ).

Techniques: Construct, Binding Assay, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Activation Assay

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) The co-crystal structure of RN129 ( 28 ) with CLK3 highlighting the type II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3). ( B ) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI) built into the cryo-EM map. ( C and D ) Close-ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 (C) and LRRK2 RCKW :GZD824 (PDB: 8TZE) (D). ( E ) Superposition of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (in lighter shades) and our previously published structure of a LRRK2 RCKW :MLi-2:E11 DARPin complex (PDB: 8TXZ) (in darker shades). Only the kinase domains, which were aligned on their C-lobes, are shown. Major features of the kinase, including those that are indicators of type I and type II inhibitor binding, are shown.

Article Snippet: The LRRK2 KW (RRID: Addgene_228879) or LRRK2 RCKW (RRID: Addgene_226783) expression constructs (table S4) encoding a Tobacco Etch Virus (TEV) cleavable N-terminal His 6 -Z-tag were expressed in SF9 insect cells (RRID:CVCL_0549) after baculoviral transfection as described previously ( ).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue or light blue. The 18.5 K ∆ T m shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size and color correlates with the degree of ∆ T m shift, as indicated in the legend. ( B ) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20-K ∆ T m shift of LRRK2 KW is highlighted in red. The bubble size or color for each kinase correlates with the ∆ T m shifts, as indicated in the legend (as in A). Kinases with ∆ T m > 6 K are labeled. ( C ) Waterfall plots of the ReactionBiology 33 PanQinase screen of RN341 at 1 and 10 μM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to <22% of the control value are labeled. ( D ) Off-target validation from both screens via in cellulo nanoBRET assay in two biological replicates, error bars ± SD, EC 50 (JNK2) = 2.7 μM, EC 50 (STK10) = 1.5 μM, EC 50 (MAPK14) = 1.7 μM, EC 50 (TTK) = 3.2 μM, EC 50 (CDKL1) = 17 μM, EC 50 (CLK1) = 6.0 μM, EC 50 (JNK3) = 15 μM, EC 50 (DYRK2) ≥ 20 μM, EC 50 (SLK) > 20 μM, EC 50 (DDR2) > 20 μM, and EC 50 (STK17B) ≥ 20 μM. ( E ) Representative immunoblot from 293T cells transiently co-transfected with LRRK1 and its substrate GFP-Rab7 before treatment with a dilution series of RN277 and RN341. Lysed cells were immunoblotted for LRRK1, GFP-Rab7, phospho-Rab7 (pS72), and GAPDH. ( F ) Quantification of the GFP-pRab7/GFP-Rab7/LRRK1 ratio from three independent Western blots. Statistical analysis performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons of means. P < 0.0001 for all inhibitor concentrations versus DMSO; error bars ± SEM.

Article Snippet: The LRRK2 KW (RRID: Addgene_228879) or LRRK2 RCKW (RRID: Addgene_226783) expression constructs (table S4) encoding a Tobacco Etch Virus (TEV) cleavable N-terminal His 6 -Z-tag were expressed in SF9 insect cells (RRID:CVCL_0549) after baculoviral transfection as described previously ( ).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A and B ) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. ( C ) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( D ) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. ( E ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0049, DMSO versus MLi-2; *** P = 0.0004, DMSO versus Ponatinib; *** P = 0.0006, DMSO versus 5 μM RN277; *** P = 0.0003, DMSO versus 10 μM RN277; * P = 0.0406, DMSO versus 5 μM RN341; ** P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. ( F ) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **** P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. ( G ) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( H ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0077, WT LRRK2 DMSO versus MLi-2; * P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; * P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; **** P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.

Article Snippet: The LRRK2 KW (RRID: Addgene_228879) or LRRK2 RCKW (RRID: Addgene_226783) expression constructs (table S4) encoding a Tobacco Etch Virus (TEV) cleavable N-terminal His 6 -Z-tag were expressed in SF9 insect cells (RRID:CVCL_0549) after baculoviral transfection as described previously ( ).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic of the single-molecule in vitro motility assay. ( B ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type I inhibitor MLi-2 (5 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( C ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0007, DMSO condition; *** P = 0.0003, MLi-2 condition, one-way ANOVA with Šidák’s multiple comparisons test within drug only. ( D ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type II inhibitors Ponatinib, RN277, and RN341 (10 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( E ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type II inhibitors Ponatinib, RN277, and RN341 (10 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0003, one-way ANOVA with Šidák’s multiple comparisons test within drug only.

Article Snippet: The LRRK2 KW (RRID: Addgene_228879) or LRRK2 RCKW (RRID: Addgene_226783) expression constructs (table S4) encoding a Tobacco Etch Virus (TEV) cleavable N-terminal His 6 -Z-tag were expressed in SF9 insect cells (RRID:CVCL_0549) after baculoviral transfection as described previously ( ).

Techniques: In Vitro, Motility Assay, Concentration Assay

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: CRISPR- CLONInG : Replacement of Luciferase (Luc) on FLEx vector. (A) Schematic illustration of FLEx vector with CRISPR cut sites (red scissors) at the two junction sites flanking the undesired Luc fragment. Gray dot dashes: default backbone containing origin of replication and selection for propagation in bacterial host. (B) Luc was cut out with ctRNP (Cas9-ctRNA) complex; FRT-Neo-FRT and tdTomato were PCR-amplified from existing plasmids using primers carrying complementary Gibson overhangs from the adjacent DNA fragment and vector backbone. (C) Two new vector inserts were joined with the CRISPR-digested backbone via Gibson (HiFi) Cloning for final donor assembly. (D) Excised FLEx vector backbone (∼7.5 kb) and Luciferase (∼1.65 kb) (left); PCR amplified FRT-Neo-FRT (∼1.87 kb) and tdTomato (∼1.43 kb) (right). N.S.: non-specific bands. (E) After CRISPR- CLONInG , 14 out of 20 clones verified with PstI RE(s) diagnosis showed correct vector assembly (6 DNA fragments; black arrow); 3 clones validated for sequence integrity. Resolved on 0.9% agarose gel.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Clone Assay, Luciferase, Plasmid Preparation, Selection, Amplification, Sequencing, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: CRISPR- CLONInG : Replacement of partial cargo sequence (Cre-comp) with the desired donor sequence on AAV vector (Addgene #60229). (A) Schematic illustration of AAV vector with CRISPR cut sites (red scissors) at two ends of the Cre-comp segment. Guides (AAV-A and AAV-B) with high on-target scores were selected. (B) Cre-comp was cut out with ctRNP (Cas9-ctRNA) complex; donor for gene replacement (containing 15 bp AA replacement sequence, noted as ‘R’, sandwiched by HA) flanked with complementary Gibson overhangs of the adjacent AAV backbone was PCR-amplified from custom gene synthesized plasmid. (C) Assembled AAV-v1: donor template cloned into the customized AAV backbone via Gibson (HiFi) Assembly. (D) Excised AAV vector backbone (∼3.63 kb) and Cre-comp (∼2.73 kb) (left); PCR amplified donor template (∼0.8 kb) with Gibson overhangs (right). (E) After CRISPR- CLONInG , 15 out of 16 clones showed correct vector assembly, confirmed by BbsI RE(s) diagnosis (two DNA fragments; black arrow); 3 clones further validated by Sanger sequencing. Resolved on 0.9% agarose gel.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Clone Assay, Sequencing, Plasmid Preparation, Amplification, Synthesized, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: Cloning duo-guides into AAV-v1 vector by exploiting built-in cloning site (originally designed for one sgRNA). Gene editing (AA changes at PSEN1 gene) in N2A cell line. (A) (top) Schematic AAV-v1 construct: blue dotted line zooming out partial sequences of U6 and sgRNA cloning site. (middle) Type IIS RE (SapI) digest creates two unique 5’-overhangs (GGT vs. GTT). (bottom) Showing gene synthesized plasmid carrying duo-sgRNA cassette sequence (Spacer-W+tracrRNA+U6+spacer-X) with complementary overhangs, flanked by uniquely positioned SapI sites, which upon SapI digestion renders two complementary 5’-overhangs (ACC vs. AAC; red dotted line) for cloning. (B) Schematic of the final construct AAV-v2. (C) (top) CRISPR guides (W and X) target sites (purple scissors) on PSEN1 exon 10 region. (bottom) AA replacement donor (R) integrated at the genomic target after rAAV-v2 transduction. (D) PSEN1 exon 10 sequence shown (WT vs. R: 3/5 AA changes - three amino acid codons yellow highlighted). Red arrow: SpCas9 guides (W and X) cutting sites; chromatograms showing WT, mutant (R) and hemizygous (R+indel); green dotted rectangle encompassing the 3/5 AA changes within a 15-nt range.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: Clone Assay, Plasmid Preparation, Construct, Synthesized, Sequencing, CRISPR, Transduction, Mutagenesis

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: Validation of lssDNA acquired by CRISPR- CLIP . (A) Cpf1 & WT Cas9 digested the entire plasmid (∼4.9 kb, as shown in ) into ∼2.7 kb and ∼2.2 kb DNA fragments (lane b); acquired lssDNA (∼2.2 kbase) resolved around 1.2-1.3 kb in size $ (lane d, arrow). (B) Control: the uncut plasmid (lane a); control: as described in (A) (lane b); Cpf1 and WT Cas9, along with additional BamHI digested the plasmid into three fragments: BamHI cleaved the ∼2.2 kb dsDNA template into ∼1.2 kb and ∼1 kb, while ∼2.7 kb default plasmid remained intact (lane c); BamHI digestion did not cleave the lssDNA despite bearing a BamHI cut site (lane e, arrow) vs. lssDNA without BamHI digestion (lane d); all resolve in 0.9% agarose gel electrophoresis. (C) Multiple reverse primers (red bent arrow) were used for Sanger sequencing validation of the acquired lssDNA (sense DNA, in this case). $ The lssDNA may not resolve at the exact predicted size (1.1 kb in this case), depending on certain factors, such as buffer condition and potential secondary structure formation due to sequence composition, in which case additional DGLB treatment on acquired lssDNA could aid in size separation with more precise outcomes. The integrity of the procured lssDNA is considered of good quality as long as the majority of the lssDNA resolved close to the predicted size.

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing

Journal: bioRxiv

Article Title: New Additions to the CRISPR Toolbox: CRISPR- CLONInG and CRISPR- CLIP for Donor Construction in Genome Editing

doi: 10.1101/746826

Figure Lengend Snippet: CRISPR- CLIP with add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo-PAM. (A) The dsDNA template carrying lssDNA # cassette is flanked with duo PAM-A (upstream end) and duo-PAM-B (downstream end), anchored in the default plasmid. (B) Each duo-PAM tag contains 23-bp sequence with PAMs for Cpf1 and Cas9 placed at respective 5’ ends of each DNA strand, plus a constant Cpf1 spacer sequence. Duo-PAM-A and -B are assigned with two distinct Cpf1 spacer sequences to enable suitable Cas types to make exclusive incisions on the plasmid at both ends of lssDNA junction sites. Of note, Cas9 spacer sequence is variable and subject to lssDNA donor; Cpf1 and Cas9 incisions on the duo-PAM tags will each remove 4 nts (end sequences of HA) from the lssDNA donor, which merely results in trivial variation in HA length. (C) To procure the sense ssDNA (top strand) as the lssDNA donor, choose Cas9n to cut at duo PAM-A end while choosing Cpf1 to cut at duo PAM-B end. (D) To procure the antisense ssDNA (bottom strand) as the lssDNA donor, choose Cpf1 to cut at the duo PAM-A, while using Cas9n to cut at the duo-PAM-B. (E) Upon DGLB treatment, C and D respectively yielded 3 stand-alone ssDNA units of distinct sizes resolved in 0.9% agarose gel electrophoresis. # Belongs to a locus different from the case shown in and .

Article Snippet: N2A cell (Neuro-2a/Cas9-Rosa26-Neo, GeneCopoeia, cat# SL511) was cultured in medium, comprising 44% DMEM (ATCC, cat# 30-2002), 50% Opti-MEM (Life Technologies, cat# 51985-034) with 5% FBS (Gemini, cat# 100-500) and 1% Penicillin-Streptomycin (Millipore Sigma, cat# P4333) in a 37 ℃ humid incubator with 5% CO 2 .

Techniques: CRISPR, Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis