sgrna scaffold vector  (Addgene inc)

Journal: Cell Insight

Article Title: Loop engineering of AtCas9 for effective and broad genome editing

doi: 10.1016/j.cellin.2025.100286

Figure Lengend Snippet: Z7 enhances DNA binding and cleavage activity of AtCas9 at low magnesium condition. (a) In vitro cleavage assay of WT and Z7 proteins with different concentrations of Mg 2+ (0.5, 1, 2, 5 mM). Three target loci ( Spacer-21a , CTNNB1-10g , and VEGFA-11g ) were tested. (b) Temperature-dependent cleavage efficiency of WT and Z7. (c) Thermal shift analysis of melting temperatures of the AtCas9 proteins. (d-e) EMSA analysis of binding affinities between Cas9 variants and sgRNA (d), or Cas9 RNP complexes with DNA substrates (e), under the indicated magnesium concentrations.

Article Snippet: A U6 promoter-driven AtCas9 sgRNA mammalian expression plasmid (designated Gcl203) was created by replacing the sgRNA scaffold in the gRNA_cloning Vector (Addgene plasmid 41824) with the AtCas9 sgRNA optimized scaffold.

Techniques: Binding Assay, Activity Assay, In Vitro, Cleavage Assay

Journal: Cell Insight

Article Title: Loop engineering of AtCas9 for effective and broad genome editing

doi: 10.1016/j.cellin.2025.100286

Figure Lengend Snippet: Structure-guided protein engineering improves the editing efficiency of AtCas9 . (a) Schematic representation of sgRNA: DNA target recognition by AtCas9 variants. Residue substitutions in the E535 and E5116 variants are highlighted. (b) Adenine base editing efficiencies of AtCas9 mutants at C-myc-41g and CTNNB1-10g target sites. (c) Solvent-accessible surface area (SASA) analysis of interactions between different AtCas9 variants and nucleic acids, reflecting changes in protein–nucleic acid interface accessibility. (d) (left) A-to-G editing efficiencies of wildtype and AtCas9 E5116 mutant. (right) Median fold change of editing efficiency. Data were normalized to WT AtCas9-ABE. For (b) and (d), data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by unpaired t -test (∗ p ​< ​0.033, ∗∗∗ p ​< ​0.001). Each dot represents one biological experiment.

Article Snippet: A U6 promoter-driven AtCas9 sgRNA mammalian expression plasmid (designated Gcl203) was created by replacing the sgRNA scaffold in the gRNA_cloning Vector (Addgene plasmid 41824) with the AtCas9 sgRNA optimized scaffold.

Techniques: Drug discovery, Residue, Solvent, Mutagenesis

Journal: bioRxiv

Article Title: An optimised method for generation of murine CAR-T cells by CRISPR/Cas9

doi: 10.1101/2025.11.29.689298

Figure Lengend Snippet: A) Mouse splenic T cells were isolated, activated for 48 hours then electroporated with a Cas9 RNP targeting TRAC +/- promoter-less homology directed repair templates (HDRT) +/- inhibitors of alternative double strand break repair mechanisms. HDRTs were either dsDNA with truncated cas9 target sites (tCTS) to aid localisation or AAVs with full CTS to reduce vector concatenation. Inhibitors were removed after 24 hours by media change and editing efficiency determined by flow cytometry. Spleens from up to 5 animals of the same strain were used (3 for C57Bl6TacN 3 for FVB and 5 for 129SvJ). Data in this figure are for C57Bl6TacN B) Representative flow cytometry plots, gated on single live cells. C) Cell viability by flow cytometry, gated on single cells. Data are means of technical replicates. N = 2 for dsDNA and n=3 for AAV D) Knock-in efficiency normalised to knock-out efficiency (GFP+ cells as percentage of CD3-).

Article Snippet: For vectors including an sgRNA expression cassette, geneblocks with either U6 or M11 promoters upstream of the Cas9 sgRNA scaffold, TRAC guide and polyA signal were used. pAdDeltaF6 was a gift from James M. Wilson (Addgene plasmid # 112867).

Techniques: Isolation, Plasmid Preparation, Flow Cytometry, Knock-In, Knock-Out

Journal: bioRxiv

Article Title: An optimised method for generation of murine CAR-T cells by CRISPR/Cas9

doi: 10.1101/2025.11.29.689298

Figure Lengend Snippet: A) AAV HDRT template with polycistronic construct (GFP and miniature membrane bound Luciferase) and sgRNA under either U6 or M11 (H1-7SK hybrid) promoters B) Splenic T cells from transgenic cas9 mice were activated with dynabeads and transduced with the specified MOI of AAV after 48 hours in 1uM AZD7468 and 3uM PolQi2. Percentage of cells expressing GFP above baseline level were determined by flow cytometry 5 days after transduction. Data are mean of 3 technical replicates with standard errors. C) Cas9 mouse T cells were activated with either CD3/CD28 dynabeads or platebound anti-CD3 and soluble anti-CD28 transduced at an MOI 1E5 with a U6 containing AAV at either 24 36 or 48 hours of activation. GFP positivity was assessed by flow cytometry 5 days post transduction. Data are mean of 3 technical replicates with standard errors

Article Snippet: For vectors including an sgRNA expression cassette, geneblocks with either U6 or M11 promoters upstream of the Cas9 sgRNA scaffold, TRAC guide and polyA signal were used. pAdDeltaF6 was a gift from James M. Wilson (Addgene plasmid # 112867).

Techniques: Construct, Membrane, Luciferase, Transgenic Assay, Transduction, Expressing, Flow Cytometry, Activation Assay

Journal: bioRxiv

Article Title: An optimised method for generation of murine CAR-T cells by CRISPR/Cas9

doi: 10.1101/2025.11.29.689298

Figure Lengend Snippet: Cas9 mouse T cells were isolated and activated for 48 hours with soluble anti-CD28 and platebound anti-CD3. Cells were then transduced with AAV at MOI 1E5 in the presence of 1uM AZD7468 and 3uM POlQi2 for 24 hours before expansion in fresh media with IL2 IL7 and IL15. A) Schematic of CAR HDRT constructs used. All constructs had the same 300bp homology arms to the mouse Trac locus, but differed in their marker genes, CAR constructs and the presence of flanking Cas9 target sites (CTS). The marker genes RQR8 and RMR8 and the CARs targeting GD2 or human B7H3 with or without the degron tag, iTag2, are as described in the materials and methods. B) Representative flow cytometry 5 days post AAV transduction. Colour axis on right hand plot represents the degree of CAR positivity. Cells positive for CD3ε and CAR are presumed to be γδ-T cells. C) CAR expression of all constructs after extended in vitro culture. Data are mean of 2 technical replicates. UT = untransduced D) Expansion post transduction. Data are from 3 pooled spleens transduced at an MOI 1x10^5 with JA131 or JA132. E) 24 hour co-culture of GD2 CAR (JA131) or B7H3 CAR (JA132) with 9464D expressing either GD2 or human B7H3. Data mean of are 2 independent replicates performed in technical triplicate. Target cell killing was calculated relative to the mean cell number in target only wells. F) Flow cytometry assessment of cell viability and cytotoxicity for JA132 transduced cells following cryopreservation in different cryopreservation media compared to the same cells kept in culture for less than 2 weeks. Cytotoxicity assay is as in E but normalised to target cells in wells with untransduced T cells. Data are 2 technical replicates.

Article Snippet: For vectors including an sgRNA expression cassette, geneblocks with either U6 or M11 promoters upstream of the Cas9 sgRNA scaffold, TRAC guide and polyA signal were used. pAdDeltaF6 was a gift from James M. Wilson (Addgene plasmid # 112867).

Techniques: Isolation, Transduction, Construct, Marker, Flow Cytometry, Expressing, In Vitro, Co-Culture Assay, Cytotoxicity Assay

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Western blot of protein extracts from HEK293FT cells transiently expressing different Cas13 effectors in one-vector format. (B) Immunocytochemistry of Cas13 proteins showing localization and expression. Scale bar, 20 µm. (C-D) Surviving cell number after puromycin selection for T47D (C) or LNCaP (D) cells infected by indicated one-vector Cas13 lentiviruses, mean ± SEM with n = 3. (E) Cell viability analysis for HEK293FT cells with transient transfection of indicated one-vector lentiviral constructs, mean ± SEM with n = 3. Vector: lentiv2-w/o Cas9. (F) Cell viability analysis for HEK293FT cells during lentivirus production by transient transfection of one-vector Cas13 along with packaging plasmids, mean ± SEM with n = 3. (G-I) Assessment of surviving cell number (G), lentiviral RNA (H) and integrated lentiviral DNA (I) in A549 cells infected with indicated lentiviruses at low MOI. (J-L) Assessment of surviving cell number (J), lentiviral RNA (K) and integrated lentiviral DNA (L) in A549 cells infected with indicated lentiviruses containing different crRNAs. (M) Evaluation of lentiviral defect for one-vector Cas13 systems under pHAGE-EF1α-puro plasmid backbone.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Western Blot, Expressing, Plasmid Preparation, Immunocytochemistry, Selection, Infection, Transfection, Construct

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic of RNA interactome and transcriptome analysis transiently transfected with indicated constructs in HEK293FT cells. Cas13b-crNT indicates a one-vector Cas13b system containing a non-targeting (NT) crRNA. Vector: lentiv2-w/o Cas9. (B) The number of filtered strong RIP binding peaks (pileup > 15; fold enrichment > 4) in different samples. (C) Loci and feature distribution of RIP-seq peaks for indicated samples. (D) Top enriched motifs among RIP-seq peaks for indicated samples. (E) Top GO categories enriched among RIP-seq peak-associated genes across different samples. (F) The number of differentially expressed genes that were up-regulated or down-regulated for indicated samples compared to vector control. (G-H) Top GO categories enriched among up- (G) or down-regulated (H) differentially expressed genes. (I) Venn diagrams representing overlaps of up- (left) or down-regulated (right) genes in Cas13b or Cas13b-crNT groups, versus vector.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Transfection, Construct, Plasmid Preparation, Binding Assay

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A-B) Overlap (A) and heatmap (B) of genes associated with strong Cas13-bound peaks over the transcriptome. Vector: lentiv2-w/o Cas9. (C) Viral process-related gene ontology (GO) categories enriched among Cas13-associated peaks. (D) Top consensus Cas13-associated RNA gene transcripts with viral process-related genes denoted by red asterisk. (E-F) Overlap (E) and heatmap (F) of differentially expressed genes in Cas9-or Cas13-expressing HEK293FT cells. (G) Viral process-related GO categories enriched among Cas9- or Cas13-upregulated genes. (H-I) Volcano plot (H) and top 5 enriched GO categories (I) of down- and up-regulated differentially expressed genes, Cas13b-crNT versus Cas13b. See also ; Tables S1, S2 and S3.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Sequence alignment of catalytic domains for Cas13a derived from different bacteria strains. The conserved functional residues are shown in red star or by black arrow. (B) Coomassie blue staining showing purified Cas9 and Cas13a proteins. (C) Cis RNA cleavage by indicated Cas13a variants during in vitro assay. Red arrow indicates the band position of on-target RNA or crRNA. (D-E) Trans RNA cleavage by indicated Cas13a variants during in vitro assay. Results are shown by either fluorescence signal value (D) or direct visualization under blue light illuminator (E). a.u., arbitrary unit. (F) Pre-crRNA cleavage by indicated Cas13a variants during in vitro assay. Red arrows indicate band positions of intact and different cleavage patterns of RNA with structural schematic shown in right. (G) The number of differentially expressed genes that were up-regulated or down-regulated among Cas13 variants compared to vector control. (H-I) Top functional categories enriched among up- (H) or down-regulated (I) differentially expressed genes for indicated samples. (J) RNA expression change by qPCR for indicated genes upon transient transfection with indicated constructs.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Sequencing, Derivative Assay, Functional Assay, Staining, Purification, In Vitro, Fluorescence, Plasmid Preparation, RNA Expression, Transfection, Construct

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) RIP-seq tracks of indicated genes for Cas13a. Red rectangle denotes RNA fragment used for in vitro cleavage assay. (B) Heatmap showing RNA expression pattern of indicated genes. (C) In vitro RNA cleavage for indicated RNA fragments by Cas13a-WT or Cas9. EGFP serves as an irrelevant control. (D) In vitro RNA cleavage assay for indicated RNA fragments by Cas13a variants. See also ; Tables S1, S2 and S3.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: In Vitro, Cleavage Assay, RNA Expression