hcas9 expression vector  (Addgene inc)

Journal: BMC Biology

Article Title: Exonuclease editor promotes precision of gene editing in mammalian cells

doi: 10.1186/s12915-024-01918-w

Figure Lengend Snippet: Characterization of the EXO editor system. A The schematic diagram illustrates the EXO editor system, where Cas9 cuts both strands of DNA to create DSBs. Following this, the exonuclease initiates a long-distance resection from the 5′ to the 3′ end of the DNA, which results in the formation of a lengthy 3′ single-stranded overhang. This overhang is essential for the subsequent process of strand invasion. B Representation of constructs of the EXO editors (CXE, EXC, CTE, and ETC) and controls (Cas9, E4, and E1B). C HDR efficiencies detected by flow cytometry in HEK293 cells with different EXO editors or controls at h AAVS1 and h GAPDH loci. D Indel frequencies quantified by amplicon deep sequencing in HEK293 cells with different EXO editors or controls at h AAVS1 and h GAPDH loci. E Relative HDR: indel ratio normalized to the Cas9 at h AAVS1 and h GAPDH loci. F Strategy for insertion of EGFP-expressing cassette into the h Rosa26 locus in HEK293 cells. The h Rosa26 -sgRNA targeted sequence is highlighted in red, with the PAM sequence highlighted in green. The targeting vector consists of 5′- and 3′-homology arms that flank the SA-EGFP-polyA cassette. G The bar plot illustrates the efficiencies of HDR induced by Cas9 or CXE at the h Rosa26 locus of the HEK293 cells. The efficiencies of HDR were quantified by the percentage of EGFP-positive cells. Flow cytometry analysis was conducted on the samples 3 days post-transfection. Frequencies of HDR or indels (mean ± s.d.) were calculated from three independent experiments ( n = 3) in C , D , E , and G . Independent experiments were performed in triplicate and data were shown as black dots. P values were obtained using unpaired t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , no significant

Article Snippet: All of them were separately cloned into Cas9 expression vector backbone (Addgene, #41815).

Techniques: Construct, Flow Cytometry, Amplification, Sequencing, Expressing, Plasmid Preparation, Transfection

Journal: BMC Biology

Article Title: Exonuclease editor promotes precision of gene editing in mammalian cells

doi: 10.1186/s12915-024-01918-w

Figure Lengend Snippet: EXO editor inhibits NHEJ locally but not globally. A The bar plot illustrates the efficiencies of NHEJ induced by Cas9 or CXE at the EGFP locus of the HEK293-EGFP cells. The efficiencies of NHEJ were quantified by the percentage of EGFP-negative cells. B Stacked bar plot illustrates the efficiencies of NHEJ induced by Cas9 or CXE at three endogenous genes ( DMD , LMNA , and TP53 ) of the HEK293 cells. C , D Fluorescent images ( C ) and representative flow cytometry plots ( D ) of HEK293-EGFP cells co-electroporated Cas12a, Cas12a+CXE, or AXE with Cas12a-EGFP-sgRNA. Scale bar, 200 μm. E The bar plot illustrates the efficiencies of NHEJ induced by Cas12a, Cas12a+CXE, or AXE at the EGFP locus in HEK293 cells. The efficiencies of NHEJ were quantified by the percentage of EGFP-negative cells. F NHEJ editing frequencies induced by Cas12a, Cas12a+CXE, or AXE at four endogenous loci ( APOE , B2M , CYPE , and HMGA1 ) in HEK293 cells. Values and error bars in A , E , and F indicate the mean ± s.d. from three independent experiments ( n = 3). P values were obtained using unpaired t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , no significant

Article Snippet: All of them were separately cloned into Cas9 expression vector backbone (Addgene, #41815).

Techniques: Flow Cytometry

Journal: BMC Biology

Article Title: Exonuclease editor promotes precision of gene editing in mammalian cells

doi: 10.1186/s12915-024-01918-w

Figure Lengend Snippet: Precise exon insertion in DMD patient-derived hiPSCs with exon 51 deletion. A Workflow of screening hiPSCs colonies with E51 insertion through positive-negative selection. B Schematic of donor DNA and detailed procedures of positive-negative selection. C PCR-based genotyping of mCherry-positive colonies after first-round editing with Cas9 or CXE followed by picking up single-cell colonies. D PCR-based genotyping of mCherry-negative colonies after second-round editing with Cre recombinase followed by picking up single-cell colonies. E Fluorescent images and representative flow cytometry plots of indicated hiPSCs. Scale bar, 400 μm

Article Snippet: All of them were separately cloned into Cas9 expression vector backbone (Addgene, #41815).

Techniques: Derivative Assay, Selection, Flow Cytometry

Journal: BMC Biology

Article Title: Exonuclease editor promotes precision of gene editing in mammalian cells

doi: 10.1186/s12915-024-01918-w

Figure Lengend Snippet: DMD iPSC-derived cardiomyocytes restore the open reading frame of dystrophin after Cas9 or CXE-mediated genome editing. A Illustration of exon 51 insertion in a mutant DMD , leading to restoration of the reading frame. Red asterisk indicates termination codon. B Bright-field images of the indicated DMD iPSC-derived cardiomyocytes. Scale bar, 400 μm. C RT-PCR analysis of dystrophin mRNA transcripts in Cas9 or CXE-edited iPSC-derived cardiomyocytes. The 514-bp band in the WT and corrected lane is the dystrophin transcript from exons 49 to 52. The 281-bp band in the uncorrected lane is the dystrophin transcript from exons 49 to 52 with exon 51 deletion. D Representative chromatograms of RT-PCR products shown in C . Dashed line denotes exon boundary. E An amplicon containing junctions between exons 50 and 52 was amplified from dystrophin cDNA and analyzed by HTS. Sashimi plots show the coverage of each exon. F Details of reads coverage and exon junctions. Dashed red line denotes exon boundary

Article Snippet: All of them were separately cloned into Cas9 expression vector backbone (Addgene, #41815).

Techniques: Derivative Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: BMC Biology

Article Title: Exonuclease editor promotes precision of gene editing in mammalian cells

doi: 10.1186/s12915-024-01918-w

Figure Lengend Snippet: Modification profile of CXE-assistant MMEJ with paired sgRNAs. A Schematic of CXE-mediated precise deletion. Target DNA sequences contain two protospacer sequences on the opposite strands. CXE target each protospacer and generate long 3′ single-stranded overhangs on each side of DSB. The MH sequences on each side of the overhang anneal to each other. This process results in deletion of one of the MH sequences and the intervening non-homologous DNA flaps. MH sequence is indicated in yellow and the PAM is shown in red. B NHEJ editing frequencies induced by CXE with the aid of paired sgRNAs at the three endogenous genomic loci in HEK293 cells. C MH: indel ratio at CFTR , Rosa26 , and EMX1 loci in HEK293 cells. D Proportions of indicated MH sequences among total MH reads generated by Cas9 and CXE at the three endogenous loci. E Heat maps show the frequency of MH reads with different length. F Size distribution of deletions among total deletions induced by Cas9 and CXE at the three endogenous loci. P values in B and C were obtained using unpaired t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , no significant. Error bars indicated standard deviation of n = 3 biological replicates. Independent experiments were performed in triplicate

Article Snippet: All of them were separately cloned into Cas9 expression vector backbone (Addgene, #41815).

Techniques: Modification, Sequencing, Generated, Standard Deviation

Journal: BMC Biology

Article Title: Exonuclease editor promotes precision of gene editing in mammalian cells

doi: 10.1186/s12915-024-01918-w

Figure Lengend Snippet: Additive precision stimulation by CXE nickase and CXE fusion to different domains. A HDR efficiencies measured by flow cytometry in HEK293 cells treated with different Cas9 and CXE nickase at h AAVS1 and h Rosa26 loci. B Indel frequencies quantified by high-throughput sequencing in HEK293 cells treated with different Cas9 and CXE nickase at h AAVS1 and h Rosa26 loci. C Relative HDR: indel ratio normalized to the Cas9 control at h AAVS1 and h Rosa26 loci in HEK293 cells treated with different Cas9 and CXE nickase. D HDR efficiencies measured by flow cytometry in HEK293 cells treated with ECXE and DCXE at h AAVS1 , h GAPDH , and h Rosa26 loci. E Indel frequencies quantified by high-throughput sequencing in HEK293 cells treated with ECXE and DCXE at h AAVS1 , h GAPDH , and h Rosa26 loci. F Relative HDR: indel ratio normalized to the Cas9 control at h AAVS1 , h GAPDH , and h Rosa26 loci in HEK293 cells treated with ECXE and DCXE. P values in A–F were obtained using unpaired t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns , no significant. Error bars indicated standard deviation of n = 3 biological replicates. Independent experiments were performed in triplicate and data were shown as black dots

Article Snippet: All of them were separately cloned into Cas9 expression vector backbone (Addgene, #41815).

Techniques: Flow Cytometry, Next-Generation Sequencing, Control, Standard Deviation

Journal: Scientific Reports

Article Title: The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice

doi: 10.1038/s41598-023-49016-1

Figure Lengend Snippet: Establishment of IL-17RA-deficient AX cells. ( a ) Two independent IL-17RA-deficient AX lines (KO1 and KO2) were generated by lentiviral transduction using the CRISPR/Cas9 system. AX cells transduced with scramble constructs served as controls (AX-SCR). IL-17RA expression on these AX cell lines, which harbor a GFP tag, was analyzed by flow cytometry. Solid or dotted lines in histograms represent AX cells stained with anti-IL-17RA or isotype control antibody, respectively. ( b ) IL-17RA-deficient and control AX cells (Parental AX cells and AX-SCR) were cultured 24 h with or without (vehicle) IL-17A (500 ng/ml) and the number of AX cells was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay measuring absorbance at 570 nm. Data represent mean absorbance ± s.d. (n = 6, * P < 0.05). ( c ) IL-17RA knockout (middle and bottom rows) or scramble-transduced AX cells (top row) were treated with or without (vehicle) IL-17A (500 ng/ml). After 24 h, total RNA was prepared, and expression of ALP , Oc or Runx2 relative to β-actin was analyzed by quantitative real-time PCR. Data represent mean indicated gene expression relative to β-actin ± s.d. (n = 6, * P < 0.05). ( d ) IL-17RA knockout (middle and right rows) or scramble-transduced AX cells (left rows) were treated with or without (vehicle) IL-17A (500 ng/ml). After 48 h, total protein was extracted, and ALP, Oc and Runx2 levels were analyzed by western blot. The graph shows the normalization of ALP, Oc and Runx2 levels to total Actin levels (* P < 0.05, n = 3).

Article Snippet: The lentiviral pLV[2CRISPR]-hCas9:T2A:Puro-U6 expression vector (VectorBuilder, Inc., Chicago, IL, USA) containing the sequence of murine IL-17RA gRNAs (two independent sequences) or scramble gRNA were used. gRNA sequences were as follows.

Techniques: Generated, Transduction, CRISPR, Construct, Expressing, Flow Cytometry, Staining, Cell Culture, Knock-Out, Real-time Polymerase Chain Reaction, Western Blot