t7e1 endonuclease  (New England Biolabs)


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    Name:
    T7 Endonuclease I
    Description:
    T7 Endonuclease I 1 250 units
    Catalog Number:
    M0302L
    Price:
    282
    Category:
    Other Endonucleases
    Size:
    1 250 units
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    New England Biolabs t7e1 endonuclease
    T7 Endonuclease I
    T7 Endonuclease I 1 250 units
    https://www.bioz.com/result/t7e1 endonuclease/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t7e1 endonuclease - by Bioz Stars, 2021-09
    99/100 stars

    Images

    1) Product Images from "Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation"

    Article Title: Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2014.57

    Analysis of LTC-IC. CD34+ cells were transduced with HD-bGlob and HD-ZFN at the indicated MOIs. Three days later, cells were transferred to LTC-IC medium and cultured for 5 weeks. A total of 3,000 LTC-IC cells were then plated in methylcellulose supplemented with growth factors and cytokines. Two weeks later, colonies were counted. Cells from all colonies per plate were combined, genomic DNA was isolated, and subjected to T7E1 nuclease assay. ( a,b ) Numbers of colonies per plate for donor A and B, respectively. There was no difference in the ratio of BFU-E and CFU-GM colonies in the different groups. N = 3 plates. n.s., nonsignificant ( P > 0.05), ** P
    Figure Legend Snippet: Analysis of LTC-IC. CD34+ cells were transduced with HD-bGlob and HD-ZFN at the indicated MOIs. Three days later, cells were transferred to LTC-IC medium and cultured for 5 weeks. A total of 3,000 LTC-IC cells were then plated in methylcellulose supplemented with growth factors and cytokines. Two weeks later, colonies were counted. Cells from all colonies per plate were combined, genomic DNA was isolated, and subjected to T7E1 nuclease assay. ( a,b ) Numbers of colonies per plate for donor A and B, respectively. There was no difference in the ratio of BFU-E and CFU-GM colonies in the different groups. N = 3 plates. n.s., nonsignificant ( P > 0.05), ** P

    Techniques Used: Transduction, Cell Culture, Isolation, Nuclease Assay

    Structure and functional analysis of an HD-Ad5/35 vector expressing a globin LCR-specific TALEN. ( a ) Target site of TALEN. Shown is the structure of the globin LCR with DNase hypersensitivity sites HS1 to HS5. The lower panel shows the 5′ sequence of the HS2 target site labeled by a horizontal arrow. The lines above and below the sequence indicate the binding sites of the two TALEN subunits, respectively. The vertical bold arrow marks the TALEN cleavage site. ( b ) Structure of the HD-Ad5/35.TALENmiR (HD-TALEN) genome. In analogy to the ZFN vector, the two TALEN subunits were linked through a 2A peptide at the 3′ end to the miR183/218 target sequence-containing 3′UTR. The N-terminus of TALEN (1) contained an influenza hemagglutinine (HA) tag. ( c ) Expression of TALEN in MO7e cells. Cells were infected at an MOI of 1,000 vp/cell, and cell lysates were analyzed by western blot with antibodies specific for HA-tag. ( d ) T7E1 nuclease assay analysis. Genomic DNA was isolated from MO7e cells 48 hours after infection at an MOI of 10 3 , 2 × 10 3 vp/cell and subjected to PCR using globin LCR H2-specific primers. The expected length of PCR products is 608, 434, and 174 bp. MOI, multiplicity of infection; vp, viral particle; ZFN, zinc-finger nuclease.
    Figure Legend Snippet: Structure and functional analysis of an HD-Ad5/35 vector expressing a globin LCR-specific TALEN. ( a ) Target site of TALEN. Shown is the structure of the globin LCR with DNase hypersensitivity sites HS1 to HS5. The lower panel shows the 5′ sequence of the HS2 target site labeled by a horizontal arrow. The lines above and below the sequence indicate the binding sites of the two TALEN subunits, respectively. The vertical bold arrow marks the TALEN cleavage site. ( b ) Structure of the HD-Ad5/35.TALENmiR (HD-TALEN) genome. In analogy to the ZFN vector, the two TALEN subunits were linked through a 2A peptide at the 3′ end to the miR183/218 target sequence-containing 3′UTR. The N-terminus of TALEN (1) contained an influenza hemagglutinine (HA) tag. ( c ) Expression of TALEN in MO7e cells. Cells were infected at an MOI of 1,000 vp/cell, and cell lysates were analyzed by western blot with antibodies specific for HA-tag. ( d ) T7E1 nuclease assay analysis. Genomic DNA was isolated from MO7e cells 48 hours after infection at an MOI of 10 3 , 2 × 10 3 vp/cell and subjected to PCR using globin LCR H2-specific primers. The expected length of PCR products is 608, 434, and 174 bp. MOI, multiplicity of infection; vp, viral particle; ZFN, zinc-finger nuclease.

    Techniques Used: Functional Assay, Plasmid Preparation, Expressing, Sequencing, Labeling, Binding Assay, Infection, Western Blot, Nuclease Assay, Isolation, Polymerase Chain Reaction, Zinc-Fingers

    Transduction studies with HD-Ad5/35.ZFNmiR. ( a ) Vector genome structure. The two ZFN subunits are linked trough a self-cleaving viral 2A peptide. The ZFN coding sequence is upstream of miR-183/218 target sites and 3′UTR. Both ZFN subunits are transcribed from the EF1a promoter. In CD34+ cells, the mRNA will not be degraded, and a polyprotein will be expressed which will subsequently be cleaved into the two ZFN subunits at the 2A peptide. ( b , d ) Expression of ZFN protein in ( b ) MO7e cells or ( d ) CD34+ cells after transduction with the HD-Ad5/35.ZFNmiR vector (HD-ZFN) at the indicated MOIs. Cells were harvested 48 hours later, and cell lysates were analyzed by western blot with antibodies against the Fok1 domain. Actin B is used as loading control. ( c,e ) T7E1 nuclease assay. Genomic DNA from transduced ( c ) MO7e cells or ( e ) CD34+ cells was subjected to a PCR assay based on a T7E1 nuclease that detects mutations. 11 PCR products were separated by polyacrylamide gel electrophoresis. Bands that correspond to disrupted ccr5 alleles are marked by arrows. The expected size of cleavage products is 141 and 124 bp. The numbers below the lanes indicate the % of disrupted ccr5 alleles. Studies were done with CD34+ cells from donor A. MOI, multiplicity of infection; ZFN, zinc-finger nuclease.
    Figure Legend Snippet: Transduction studies with HD-Ad5/35.ZFNmiR. ( a ) Vector genome structure. The two ZFN subunits are linked trough a self-cleaving viral 2A peptide. The ZFN coding sequence is upstream of miR-183/218 target sites and 3′UTR. Both ZFN subunits are transcribed from the EF1a promoter. In CD34+ cells, the mRNA will not be degraded, and a polyprotein will be expressed which will subsequently be cleaved into the two ZFN subunits at the 2A peptide. ( b , d ) Expression of ZFN protein in ( b ) MO7e cells or ( d ) CD34+ cells after transduction with the HD-Ad5/35.ZFNmiR vector (HD-ZFN) at the indicated MOIs. Cells were harvested 48 hours later, and cell lysates were analyzed by western blot with antibodies against the Fok1 domain. Actin B is used as loading control. ( c,e ) T7E1 nuclease assay. Genomic DNA from transduced ( c ) MO7e cells or ( e ) CD34+ cells was subjected to a PCR assay based on a T7E1 nuclease that detects mutations. 11 PCR products were separated by polyacrylamide gel electrophoresis. Bands that correspond to disrupted ccr5 alleles are marked by arrows. The expected size of cleavage products is 141 and 124 bp. The numbers below the lanes indicate the % of disrupted ccr5 alleles. Studies were done with CD34+ cells from donor A. MOI, multiplicity of infection; ZFN, zinc-finger nuclease.

    Techniques Used: Transduction, Plasmid Preparation, Sequencing, Expressing, Western Blot, Nuclease Assay, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Infection, Zinc-Fingers

    ccr5 Gene knock out in NOD/SCID repopulating cells. ( a ) Study design. Cryoconserved CD34+ cells from donor A were cultured overnight under low cytokine concentration conditions and transduced with HD-bGlob or HD-ZFN at an MOI of 5,000 vp/cell for 24 hours. Cells were then washed and transplanted into sublethally irradiated NOG mice. Six weeks later, animals were euthanized, and bone marrow cells, splenocytes, and PBMC were collected. The percentage of human cells in collected cells was measured by flow cytometry for the pan-leukocyte marker CD45. Human donor cells were purified by magnetic-activated cell sorting (MACS) using beads conjugated with anti-human CD45 antibodies. CD45+ cells were used for the T7E1 nuclease assay. ( b ) Engraftment rate based on the percentage of human CD45+ cells in total cells from bone marrow, spleen, and PBMCs. N = 3. ( c ) Number of colonies from MACS isolated human CD45+ from bone marrow cells in the bone marrow of transplanted mice. N = 3. The difference between “no Ad” and “HD-ZFN” is not significant ( P = 0.061) ( d ) Analysis of ccr5 gene disruption in human CD45+ cells from bone marrow of transplanted mice. vp, viral particle; ZFN, zinc-finger nuclease.
    Figure Legend Snippet: ccr5 Gene knock out in NOD/SCID repopulating cells. ( a ) Study design. Cryoconserved CD34+ cells from donor A were cultured overnight under low cytokine concentration conditions and transduced with HD-bGlob or HD-ZFN at an MOI of 5,000 vp/cell for 24 hours. Cells were then washed and transplanted into sublethally irradiated NOG mice. Six weeks later, animals were euthanized, and bone marrow cells, splenocytes, and PBMC were collected. The percentage of human cells in collected cells was measured by flow cytometry for the pan-leukocyte marker CD45. Human donor cells were purified by magnetic-activated cell sorting (MACS) using beads conjugated with anti-human CD45 antibodies. CD45+ cells were used for the T7E1 nuclease assay. ( b ) Engraftment rate based on the percentage of human CD45+ cells in total cells from bone marrow, spleen, and PBMCs. N = 3. ( c ) Number of colonies from MACS isolated human CD45+ from bone marrow cells in the bone marrow of transplanted mice. N = 3. The difference between “no Ad” and “HD-ZFN” is not significant ( P = 0.061) ( d ) Analysis of ccr5 gene disruption in human CD45+ cells from bone marrow of transplanted mice. vp, viral particle; ZFN, zinc-finger nuclease.

    Techniques Used: Knock-Out, Cell Culture, Concentration Assay, Transduction, Irradiation, Mouse Assay, Flow Cytometry, Cytometry, Marker, Purification, FACS, Magnetic Cell Separation, Nuclease Assay, Isolation, Zinc-Fingers

    2) Product Images from "Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression"

    Article Title: Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0187236

    Paired-nickase DSB induction by pDG462. Sox1 or Sox3 PCR followed by T7E1 assay was performed on pDG462-transfected samples. Mutations in Sox1 and Sox3 were induced by pDG462 Sox1A/Sox1B or pDG462 Sox3A/Sox3B, respectively, as indicated by the digested products after T7E1 treatment (blue arrows). Mutations were not induced by non-paired-nickase control plasmids (pDG462 Sox1A/Sox3A or pDG462 Sox1B/Sox3B). Complete figures with more independent samples can be found in S3 Fig .
    Figure Legend Snippet: Paired-nickase DSB induction by pDG462. Sox1 or Sox3 PCR followed by T7E1 assay was performed on pDG462-transfected samples. Mutations in Sox1 and Sox3 were induced by pDG462 Sox1A/Sox1B or pDG462 Sox3A/Sox3B, respectively, as indicated by the digested products after T7E1 treatment (blue arrows). Mutations were not induced by non-paired-nickase control plasmids (pDG462 Sox1A/Sox3A or pDG462 Sox1B/Sox3B). Complete figures with more independent samples can be found in S3 Fig .

    Techniques Used: Polymerase Chain Reaction, Transfection

    3) Product Images from "Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts"

    Article Title: Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0454-1

    Schematic representation of this study and virus production in DF-1 cells by RCAS vectors. A Overview of this study. The CRISPR/Cas9 vectors including Cas9 protein-coding sequences, tvb -targeting guide RNA and neomycin resistance genes were transfected into DF-1 cells. After G418 selection, T7E1 assays and TA cloning were performed. tvb -modified single DF-1 cells were cultured in 96-well plates, and tvb from individual DF-1 clones was sequenced. Clones were infected with ALV subgroup B produced by RCASBP-(B)-CN-EGFP vector-transfected DF-1 cells. B ALV subgroup B production in DF-1 cells. DF-1 cells transfected with RCASBP-(B)-CN-EGFP vectors expressed green fluorescent protein (GFP). Non-transfected DF-1 cells (WT) used as negative control. Scale bar = 200 µm.
    Figure Legend Snippet: Schematic representation of this study and virus production in DF-1 cells by RCAS vectors. A Overview of this study. The CRISPR/Cas9 vectors including Cas9 protein-coding sequences, tvb -targeting guide RNA and neomycin resistance genes were transfected into DF-1 cells. After G418 selection, T7E1 assays and TA cloning were performed. tvb -modified single DF-1 cells were cultured in 96-well plates, and tvb from individual DF-1 clones was sequenced. Clones were infected with ALV subgroup B produced by RCASBP-(B)-CN-EGFP vector-transfected DF-1 cells. B ALV subgroup B production in DF-1 cells. DF-1 cells transfected with RCASBP-(B)-CN-EGFP vectors expressed green fluorescent protein (GFP). Non-transfected DF-1 cells (WT) used as negative control. Scale bar = 200 µm.

    Techniques Used: CRISPR, Transfection, Selection, TA Cloning, Modification, Cell Culture, Clone Assay, Infection, Produced, Plasmid Preparation, Negative Control

    Genetic modification of tvb by CRISPR/Cas9 in DF-1 cells. A Gene structure of tvb (TNFRSF10B) and recognition sites of TVB#1 and TVB#2 CRISPR/Cas9 vectors. Blue bars indicate guide RNA recognition sites, and red bars indicate protospacer-adjacent motif (PAM) sequences. Scale bar = 1 kb. B T7E1 assay for DF-1 cells transfected with TVB#1 and TVB#2 CRISPR/Cas9 vectors. Bands cleaved by T7E1 endonuclease were seen in the experimental groups. C Sequencing analysis of transfected DF-1 cells using the TA cloning method. Grey letters indicate insertions, and grey letters with lines indicate deletions. Indel mutations and their frequencies are presented. Blue bars indicate guide RNA recognition sites, and red bars indicate PAM sequences. Wild type (WT) DF-1 cells were used as the control.
    Figure Legend Snippet: Genetic modification of tvb by CRISPR/Cas9 in DF-1 cells. A Gene structure of tvb (TNFRSF10B) and recognition sites of TVB#1 and TVB#2 CRISPR/Cas9 vectors. Blue bars indicate guide RNA recognition sites, and red bars indicate protospacer-adjacent motif (PAM) sequences. Scale bar = 1 kb. B T7E1 assay for DF-1 cells transfected with TVB#1 and TVB#2 CRISPR/Cas9 vectors. Bands cleaved by T7E1 endonuclease were seen in the experimental groups. C Sequencing analysis of transfected DF-1 cells using the TA cloning method. Grey letters indicate insertions, and grey letters with lines indicate deletions. Indel mutations and their frequencies are presented. Blue bars indicate guide RNA recognition sites, and red bars indicate PAM sequences. Wild type (WT) DF-1 cells were used as the control.

    Techniques Used: Modification, CRISPR, Transfection, Sequencing, TA Cloning

    4) Product Images from "A molecular network regulating the pro-inflammatory phenotype of human memory T lymphocytes"

    Article Title: A molecular network regulating the pro-inflammatory phenotype of human memory T lymphocytes

    Journal: Nature immunology

    doi: 10.1038/s41590-020-0622-8

    Optimization of CRISP-Cas9-mediated deletion and screening. a) As a proof-of-principle and test of efficiency, Jurkat T cells were transfected using the Neon transfection system with ribonucleoparticles of Cas9 and one single guide RNA against the TCRα chain. After three days the cells were stained for surface TCR expression, showing a loss of expression in 62% of the cells (green). Grey: mock transfected control cells. Representative of at least n=3 independent experiments. b) Schematic representation of the BHLHE40 locus with indicated the location of guide RNAs and primers used for screening of the clones. c) Optimization of screening procedure by mismatch cleavage assay. After transfection with two guide RNAs against BHLHE40 , Jurkat cells were single cell-cloned by seeding in a 384-well plate in 20% FBS. Genomic DNA was extracted from 10 clones and from the entire population (no cloning) and negative controls. The first ‘long’ PCR showed already the presence of indels in most clones (left panel) and even at the level of whole population. This was further confirmed by denaturing, reannealing and T7 endonuclease digestion (middle panel). The presence of a homozygous deletion was further confirmed using a ‘short’ PCR that cannot provide an amplification product if the region between the two guide RNAs is deleted. The highlighted Clone 7 (black box) is one of several clones in which the deletion was most likely identical on both alleles, leading to a shorter PCR product that cannot be digested by the T7 endonuclease because of the absence of mismatches. Representative of at least n=2 independent transfections and cloning procedures. d) The genomic DNA from Clone 7 was sequenced in the BHLHE40 locus region, revealing a deletion of 196 nucleotides involving part of exon 1 and exon 2, as well as the intron. e) Schematic representation of the ZC3H12D locus with indicated the location of guide RNAs and primers used for screening of the clones in . Figure 8f
    Figure Legend Snippet: Optimization of CRISP-Cas9-mediated deletion and screening. a) As a proof-of-principle and test of efficiency, Jurkat T cells were transfected using the Neon transfection system with ribonucleoparticles of Cas9 and one single guide RNA against the TCRα chain. After three days the cells were stained for surface TCR expression, showing a loss of expression in 62% of the cells (green). Grey: mock transfected control cells. Representative of at least n=3 independent experiments. b) Schematic representation of the BHLHE40 locus with indicated the location of guide RNAs and primers used for screening of the clones. c) Optimization of screening procedure by mismatch cleavage assay. After transfection with two guide RNAs against BHLHE40 , Jurkat cells were single cell-cloned by seeding in a 384-well plate in 20% FBS. Genomic DNA was extracted from 10 clones and from the entire population (no cloning) and negative controls. The first ‘long’ PCR showed already the presence of indels in most clones (left panel) and even at the level of whole population. This was further confirmed by denaturing, reannealing and T7 endonuclease digestion (middle panel). The presence of a homozygous deletion was further confirmed using a ‘short’ PCR that cannot provide an amplification product if the region between the two guide RNAs is deleted. The highlighted Clone 7 (black box) is one of several clones in which the deletion was most likely identical on both alleles, leading to a shorter PCR product that cannot be digested by the T7 endonuclease because of the absence of mismatches. Representative of at least n=2 independent transfections and cloning procedures. d) The genomic DNA from Clone 7 was sequenced in the BHLHE40 locus region, revealing a deletion of 196 nucleotides involving part of exon 1 and exon 2, as well as the intron. e) Schematic representation of the ZC3H12D locus with indicated the location of guide RNAs and primers used for screening of the clones in . Figure 8f

    Techniques Used: Transfection, Staining, Expressing, Cleavage Assay, Clone Assay, Polymerase Chain Reaction, Amplification

    5) Product Images from "Cruciform DNA Structure Underlies the Etiology for Palindrome-mediated Human Chromosomal Translocations *"

    Article Title: Cruciform DNA Structure Underlies the Etiology for Palindrome-mediated Human Chromosomal Translocations *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M400354200

    Nuclease sensitivity assay of the PATRR plasmid The original plasmid, plasmid with S1 nuclease digestion, plasmid with T7 endonuclease digestion, and plasmid with restriction enzyme digestion that cuts the plasmid only once were subjected to agarose gel electrophoresis in this order. Lanes 1–4 , psPATRR11; lanes 5–8 , pΔPATRR11; lanes 9–12 , TA cloning vector. The arrows indicate the position of the linear form of psPATRR11 ( a ), pΔPATRR11 ( b ), and TA cloning vector ( c ). S1 , S1 nuclease; T7 , T7 endonuclease; RE , restriction enzyme.
    Figure Legend Snippet: Nuclease sensitivity assay of the PATRR plasmid The original plasmid, plasmid with S1 nuclease digestion, plasmid with T7 endonuclease digestion, and plasmid with restriction enzyme digestion that cuts the plasmid only once were subjected to agarose gel electrophoresis in this order. Lanes 1–4 , psPATRR11; lanes 5–8 , pΔPATRR11; lanes 9–12 , TA cloning vector. The arrows indicate the position of the linear form of psPATRR11 ( a ), pΔPATRR11 ( b ), and TA cloning vector ( c ). S1 , S1 nuclease; T7 , T7 endonuclease; RE , restriction enzyme.

    Techniques Used: Sensitive Assay, Plasmid Preparation, Agarose Gel Electrophoresis, TA Cloning

    Two-dimensional gel electrophoresis of the PATRR plasmid A , psPATRR11 prepared by the alkaline lysis method.  B , psPATRR11 treated with topoisomerase I in the presence of various amounts of ethidium bromide.  C , topoisomerase I-treated psPATRR11 digested with T7 endonuclease.  D , topoisomerase I-treated psPATRR11 digested with S1 nuclease. Two downward-sloping curves originating from cruciform extrusion are observed at the  lower right side  on both the  A  and the  B  gels, but neither are observed in the  C  nor in the  D  gel.  Spots  at the  upper left side  on the gels originate from open circular nicked plasmids, whereas  spots  near the  center  of the gels are from nuclease-cleaved linear plasmids.
    Figure Legend Snippet: Two-dimensional gel electrophoresis of the PATRR plasmid A , psPATRR11 prepared by the alkaline lysis method. B , psPATRR11 treated with topoisomerase I in the presence of various amounts of ethidium bromide. C , topoisomerase I-treated psPATRR11 digested with T7 endonuclease. D , topoisomerase I-treated psPATRR11 digested with S1 nuclease. Two downward-sloping curves originating from cruciform extrusion are observed at the lower right side on both the A and the B gels, but neither are observed in the C nor in the D gel. Spots at the upper left side on the gels originate from open circular nicked plasmids, whereas spots near the center of the gels are from nuclease-cleaved linear plasmids.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Alkaline Lysis

    Mapping of nuclease cleavage sites in the PATRR A , mapping by digestion with restriction enzyme. S1 , S1 nuclease; T7 , T7 endonuclease; RE , restriction enzyme. B , restriction map of the PATRR-flanking region. C , mapping by sequencing. The entire PATRR is shown as a putative hairpin structure predicted by mfold software (mfold.burnet.edu.au/dna_form). The arrows indicate cleavage sites with T7 endonuclease, whereas the arrowheads indicate those with S1 nuclease. the relatively GC-rich region is indicated by a bracket .
    Figure Legend Snippet: Mapping of nuclease cleavage sites in the PATRR A , mapping by digestion with restriction enzyme. S1 , S1 nuclease; T7 , T7 endonuclease; RE , restriction enzyme. B , restriction map of the PATRR-flanking region. C , mapping by sequencing. The entire PATRR is shown as a putative hairpin structure predicted by mfold software (mfold.burnet.edu.au/dna_form). The arrows indicate cleavage sites with T7 endonuclease, whereas the arrowheads indicate those with S1 nuclease. the relatively GC-rich region is indicated by a bracket .

    Techniques Used: Sequencing, Software

    6) Product Images from "Microneedle-assisted genome editing: A transdermal strategy of targeting NLRP3 by CRISPR-Cas9 for synergistic therapy of inflammatory skin disorders"

    Article Title: Microneedle-assisted genome editing: A transdermal strategy of targeting NLRP3 by CRISPR-Cas9 for synergistic therapy of inflammatory skin disorders

    Journal: Science Advances

    doi: 10.1126/sciadv.abe2888

    Detection of NLRP3 knockout efficiency and inflammasome-related protein expression in psoriasis mice after the specified treatments. ( A ) Frequency of indel mutation detected by T7E1 assay from the skin tissues and representative Sanger sequencing results of T-A cloning from the skin tissue after dual MN patch treatment. ( B ) Immunoblot analysis of NLRP3 and other inflammasome protein expression in the dorsal skin homogenates. ( C to G ) ELISA of IL-1β (C), IL-18 (D), IL-17 (E), IL-12/23p40 (F), and TNF-α (G) production in the skin tissues of mice treated with various formulations. ( H ) H E staining of the skin tissue sections from the mice after the specified treatments (magnification, ×40 and ×100; scale bars, 500 and 200 μm, respectively). For (A) to (H), the code denotes the following: G1, normal mice without imiquimod treatment; G2, imiquimod-treated mice without therapy; G3, imiquimod-treated mice treated with blank MN patch; G4, DNCB-treated mice treated with Dex-loaded MN patch; G5, imiquimod-treated mice treated with RNP-loaded MN patch; G6, imiquimod-treated mice treated with dual-loaded MN patch; G7, imiquimod-treated mice treated with Dex cream; and G8, imiquimod-treated mice treated with tacrolimus ointment. Means ± SD, n = 6; Student’s t test, ** P
    Figure Legend Snippet: Detection of NLRP3 knockout efficiency and inflammasome-related protein expression in psoriasis mice after the specified treatments. ( A ) Frequency of indel mutation detected by T7E1 assay from the skin tissues and representative Sanger sequencing results of T-A cloning from the skin tissue after dual MN patch treatment. ( B ) Immunoblot analysis of NLRP3 and other inflammasome protein expression in the dorsal skin homogenates. ( C to G ) ELISA of IL-1β (C), IL-18 (D), IL-17 (E), IL-12/23p40 (F), and TNF-α (G) production in the skin tissues of mice treated with various formulations. ( H ) H E staining of the skin tissue sections from the mice after the specified treatments (magnification, ×40 and ×100; scale bars, 500 and 200 μm, respectively). For (A) to (H), the code denotes the following: G1, normal mice without imiquimod treatment; G2, imiquimod-treated mice without therapy; G3, imiquimod-treated mice treated with blank MN patch; G4, DNCB-treated mice treated with Dex-loaded MN patch; G5, imiquimod-treated mice treated with RNP-loaded MN patch; G6, imiquimod-treated mice treated with dual-loaded MN patch; G7, imiquimod-treated mice treated with Dex cream; and G8, imiquimod-treated mice treated with tacrolimus ointment. Means ± SD, n = 6; Student’s t test, ** P

    Techniques Used: Knock-Out, Expressing, Mouse Assay, Mutagenesis, Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Staining

    Detection of NLRP3 knockout efficiency and inflammasome-related protein expression in AD mice after the specified treatments. ( A ) Frequency of indel mutation detected by T7E1 assay from the skin tissues and representative Sanger sequencing results of T-A cloning from the skin tissue after dual MN patch treatment. ( B ) Immunoblot analysis of NLRP3 and other inflammasome protein expression in the dorsal skin homogenates. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( C ) Serum IgE levels determined by ELISA. ( D to H ) ELISA of IL-1β (D), IL-18 (E), IL-4 (F), TSLP (G), and TNF-α (H) production in the skin tissues of mice treated with various formulations. For (A) to (H), the code denotes the following: G1, normal mice without DNCB treatment; G2, DNCB-treated mice; G3, DNCB-treated mice treated with blank MN patch; G4, DNCB-treated mice treated with Dex-loaded MN patch; G5, DNCB-treated mice treated with RNP-loaded MN patch; G6, DNCB-treated mice treated with dual-loaded MN patch; G7, DNCB-treated mice treated with Dex cream; G8, DNCB-treated mice treated with tacrolimus ointment. Means ± SD, n = 6; Student’s t test, ** P
    Figure Legend Snippet: Detection of NLRP3 knockout efficiency and inflammasome-related protein expression in AD mice after the specified treatments. ( A ) Frequency of indel mutation detected by T7E1 assay from the skin tissues and representative Sanger sequencing results of T-A cloning from the skin tissue after dual MN patch treatment. ( B ) Immunoblot analysis of NLRP3 and other inflammasome protein expression in the dorsal skin homogenates. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( C ) Serum IgE levels determined by ELISA. ( D to H ) ELISA of IL-1β (D), IL-18 (E), IL-4 (F), TSLP (G), and TNF-α (H) production in the skin tissues of mice treated with various formulations. For (A) to (H), the code denotes the following: G1, normal mice without DNCB treatment; G2, DNCB-treated mice; G3, DNCB-treated mice treated with blank MN patch; G4, DNCB-treated mice treated with Dex-loaded MN patch; G5, DNCB-treated mice treated with RNP-loaded MN patch; G6, DNCB-treated mice treated with dual-loaded MN patch; G7, DNCB-treated mice treated with Dex cream; G8, DNCB-treated mice treated with tacrolimus ointment. Means ± SD, n = 6; Student’s t test, ** P

    Techniques Used: Knock-Out, Expressing, Mouse Assay, Mutagenesis, Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay

    Penetration ability and degradation of MN in vivo and improved genome-editing effects of CP/Ad-SS-GD/RNP nanoparticles. ( A ) Fluorescence images of the mouse skin recorded at different time points after insertion of the MN patch into the skin. Scale bar, 500 μm. ( B to E ) Screening targeting sequence of sgNLRP3 to optimize the genome-editing efficiency in DC2.4 cells (B) and 3T3 cells (D) and Sanger sequencing results of T-A cloning from DC2.4 cells (C) and 3T3 cells (E) after CMAX-mediated transfection. WT, wild type; N.D., not detectable. ( F and G ) T7E1 assay of indels introduced into the NLRP3 locus in DC2.4 cells (F) and 3T3 cells (G) transfected with dual CP/Ad-SS-GD/RNP and PLGA/Dex nanoparticles. Means ± SD, n = 3, Student’s t test, ** P
    Figure Legend Snippet: Penetration ability and degradation of MN in vivo and improved genome-editing effects of CP/Ad-SS-GD/RNP nanoparticles. ( A ) Fluorescence images of the mouse skin recorded at different time points after insertion of the MN patch into the skin. Scale bar, 500 μm. ( B to E ) Screening targeting sequence of sgNLRP3 to optimize the genome-editing efficiency in DC2.4 cells (B) and 3T3 cells (D) and Sanger sequencing results of T-A cloning from DC2.4 cells (C) and 3T3 cells (E) after CMAX-mediated transfection. WT, wild type; N.D., not detectable. ( F and G ) T7E1 assay of indels introduced into the NLRP3 locus in DC2.4 cells (F) and 3T3 cells (G) transfected with dual CP/Ad-SS-GD/RNP and PLGA/Dex nanoparticles. Means ± SD, n = 3, Student’s t test, ** P

    Techniques Used: In Vivo, Fluorescence, Sequencing, Clone Assay, Transfection

    7) Product Images from "Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang"

    Article Title: Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06129-w

    Optimized configuration of crRNA for highly efficient genome editing in vivo. a Scheme of the in vivo assay to determine the most efficient configuration of the crRNA. Cpf1-encoding plasmids were co-transfected with crRNA-encoding PCR amplicons into HEK-293T cells, and the Cpf1 activity was assessed by T7E1 indel assays. b Improved indel efficiency of AsCpf1 in vivo by the U-rich 3′-overhang following a 20-nt target-complementary sequence in transcribed crRNAs. This gel image is a representative result of three repeated experiments. Indel values are mean±standard deviation. c Improved indel efficiency by the 3′-end U-rich guide RNA as a unique feature of Cpf1. 3′-Proximal addition of uridinylates did not change the indel efficiency of SpCas9 in vivo. This gel image is representative of three repeated experiments. Indel values are the mean±standard deviation. d Improved indel efficiency of AsCpf1 in vivo by increased uridinylate lengths. The AsCpf1 activity was improved by the increased lengths of 3′-end uridinylates up to 8–10 mers for the chemically synthesized crRNA and up to six bases for the crRNA-encoding PCR amplicons. e Optimized 3′-end configuration of crRNA for highly efficient genome editing. Addition of the U4AU4 3′-overhang in crRNA maximized the indel efficiency of AsCpf1. * p > 0.05, ** p
    Figure Legend Snippet: Optimized configuration of crRNA for highly efficient genome editing in vivo. a Scheme of the in vivo assay to determine the most efficient configuration of the crRNA. Cpf1-encoding plasmids were co-transfected with crRNA-encoding PCR amplicons into HEK-293T cells, and the Cpf1 activity was assessed by T7E1 indel assays. b Improved indel efficiency of AsCpf1 in vivo by the U-rich 3′-overhang following a 20-nt target-complementary sequence in transcribed crRNAs. This gel image is a representative result of three repeated experiments. Indel values are mean±standard deviation. c Improved indel efficiency by the 3′-end U-rich guide RNA as a unique feature of Cpf1. 3′-Proximal addition of uridinylates did not change the indel efficiency of SpCas9 in vivo. This gel image is representative of three repeated experiments. Indel values are the mean±standard deviation. d Improved indel efficiency of AsCpf1 in vivo by increased uridinylate lengths. The AsCpf1 activity was improved by the increased lengths of 3′-end uridinylates up to 8–10 mers for the chemically synthesized crRNA and up to six bases for the crRNA-encoding PCR amplicons. e Optimized 3′-end configuration of crRNA for highly efficient genome editing. Addition of the U4AU4 3′-overhang in crRNA maximized the indel efficiency of AsCpf1. * p > 0.05, ** p

    Techniques Used: In Vivo, Transfection, Polymerase Chain Reaction, Activity Assay, Sequencing, Standard Deviation, Synthesized

    8) Product Images from "Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells"

    Article Title: Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03760-5

    Efficient generation of knockout hPSC lines using CRISPR-Cpf1. a A scheme of the experimental procedure for generating knockout hPSC lines. b Schematic of Cpf1 crRNA targeting sites at ALKBH1 and CLEC16A loci showing exon structures (green boxes), PCR amplicons (light gray boxes), and restriction sites used for PCR analysis. crRNA targeting sequences are in bold; PAM sequences are in red. c T7EI assay for crRNAs of ALKBH1 and CLEC16A in MEL1 hESCs. The Indel frequency was calculated using the expected fragments. d T7EI assay for crRNAs of ALKBH1 in H1 hESCs and hiPSCs. The Indel frequency was calculated using the expected fragments. e PCR analysis upon crRNA transfection. For ALKBH1 , two crRNAs were transfected together. Clones with gene knockout in one allele are in blue, and clones with gene knockout in two alleles are in red. More detailed description and explanation of the band pattern can be found in Supplementary Fig. 2 . f Sequencing results of the targeted allele in ALKBH1 and CLEC16A knockout hPSC lines. PAM sequences are in red. Restrictive enzyme site is in blue
    Figure Legend Snippet: Efficient generation of knockout hPSC lines using CRISPR-Cpf1. a A scheme of the experimental procedure for generating knockout hPSC lines. b Schematic of Cpf1 crRNA targeting sites at ALKBH1 and CLEC16A loci showing exon structures (green boxes), PCR amplicons (light gray boxes), and restriction sites used for PCR analysis. crRNA targeting sequences are in bold; PAM sequences are in red. c T7EI assay for crRNAs of ALKBH1 and CLEC16A in MEL1 hESCs. The Indel frequency was calculated using the expected fragments. d T7EI assay for crRNAs of ALKBH1 in H1 hESCs and hiPSCs. The Indel frequency was calculated using the expected fragments. e PCR analysis upon crRNA transfection. For ALKBH1 , two crRNAs were transfected together. Clones with gene knockout in one allele are in blue, and clones with gene knockout in two alleles are in red. More detailed description and explanation of the band pattern can be found in Supplementary Fig. 2 . f Sequencing results of the targeted allele in ALKBH1 and CLEC16A knockout hPSC lines. PAM sequences are in red. Restrictive enzyme site is in blue

    Techniques Used: Knock-Out, CRISPR, Polymerase Chain Reaction, T7EI Assay, Transfection, Clone Assay, Gene Knockout, Sequencing

    9) Product Images from "Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells"

    Article Title: Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.02.005

    Characterization of TRC1-2 Nuclease Activity in T Cells (A) Diagram of the TRC1-2 nuclease and recognition site within the TRAC locus. The TRC1-2 nuclease is a single-chain protein consisting of an N-terminal domain (N-domain) and C-terminal domain (C-domain) connected by a flexible linker. The recognition site consists of 9-bp half-sites recognized by each of the two nuclease domains, separated by a 4-bp central sequence. A broken white line in the recognition sequence denotes the overhangs generated following cleavage by the TRC1-2 nuclease. (B) A T7 endonuclease (T7E) assay was performed on mock-electroporated T cells and T cells treated with TRC1-2 nuclease on day 8 post-electroporation to confirm editing at the TRAC locus. (C) Flow cytometry staining of CD3 expression in CD4 + and CD8 + T cells on day 8 post-electroporation with TRC1-2 nuclease. Reduction of cell surface expression of CD3, a component of the TCR complex, is a functional marker of disruption of TCRα expression.
    Figure Legend Snippet: Characterization of TRC1-2 Nuclease Activity in T Cells (A) Diagram of the TRC1-2 nuclease and recognition site within the TRAC locus. The TRC1-2 nuclease is a single-chain protein consisting of an N-terminal domain (N-domain) and C-terminal domain (C-domain) connected by a flexible linker. The recognition site consists of 9-bp half-sites recognized by each of the two nuclease domains, separated by a 4-bp central sequence. A broken white line in the recognition sequence denotes the overhangs generated following cleavage by the TRC1-2 nuclease. (B) A T7 endonuclease (T7E) assay was performed on mock-electroporated T cells and T cells treated with TRC1-2 nuclease on day 8 post-electroporation to confirm editing at the TRAC locus. (C) Flow cytometry staining of CD3 expression in CD4 + and CD8 + T cells on day 8 post-electroporation with TRC1-2 nuclease. Reduction of cell surface expression of CD3, a component of the TCR complex, is a functional marker of disruption of TCRα expression.

    Techniques Used: Activity Assay, Sequencing, Generated, Electroporation, Flow Cytometry, Cytometry, Staining, Expressing, Functional Assay, Marker

    10) Product Images from "Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity"

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity

    Journal: Science Advances

    doi: 10.1126/sciadv.aay0187

    Comparison of Cas-Acr fusions to other Cas9 high-fidelity variants. Cells were cotransfected with plasmids encoding the indicated Cas9 variant and an sgRNA targeting the AAVS1, RUNX, HBB, or EMX1 locus. Following incubation for 72 hours, a T7 endonuclease assay was performed. Data are means ± SD; dots are individual data points from n = 3 independent experiments.
    Figure Legend Snippet: Comparison of Cas-Acr fusions to other Cas9 high-fidelity variants. Cells were cotransfected with plasmids encoding the indicated Cas9 variant and an sgRNA targeting the AAVS1, RUNX, HBB, or EMX1 locus. Following incubation for 72 hours, a T7 endonuclease assay was performed. Data are means ± SD; dots are individual data points from n = 3 independent experiments.

    Techniques Used: Variant Assay, Incubation

    Cas-Acr fusion design improves genome editing specificity. ( A ) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). ( B to G ) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.
    Figure Legend Snippet: Cas-Acr fusion design improves genome editing specificity. ( A ) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). ( B to G ) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.

    Techniques Used: Construct, Variant Assay, Incubation

    Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. ( A ) Schematic of a model for Cas9 genome editing. After cotransfection with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNA site , unedited target locus; DNA edited , edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. ( B ) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. ( C ) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. ( D and E ) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. ( F ) HEK 293T cells were cotransduced with 33 μl of Cas9 AAV, 33 μl of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incubated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.
    Figure Legend Snippet: Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. ( A ) Schematic of a model for Cas9 genome editing. After cotransfection with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNA site , unedited target locus; DNA edited , edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. ( B ) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. ( C ) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. ( D and E ) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. ( F ) HEK 293T cells were cotransduced with 33 μl of Cas9 AAV, 33 μl of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incubated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.

    Techniques Used: CRISPR, Cotransfection, Protein Binding, Transfection, Expressing, Modification, Activity Assay, Construct, Incubation, Plasmid Preparation

    A mathematical model of Cas-Acr action explains improved specificity and informs experimental planning. ( A ) Overview of the mathematical model of gene editing with Cas-Acr constructs. The model accounts for turnover of plasmids, sgRNA, Cas-Acr mRNA, and protein; transition between the active and inhibited states (Cas-Acr inh ); sgRNA binding (Cas-Acr:sgRNA, Cas-Acr inh :sgRNA); association with a target gene; and gene editing. ( B ) Exemplary model fits to time-resolved T7 endonuclease assay measurements using the AAVS1-targeting sgRNA and either wild-type Cas9 or the Cas-Acr variant Ins. 5 (see fig. S10 for the full set of fits). ( C ) ON- and OFF-target editing efficiencies for sgRNAs targeting the AAVS1, EMX1, RUNX1, or HEK locus are shown together with model simulations of editing efficiencies for either wild-type Cas9 or the indicated Cas-Acr variants. The model was calibrated with ON- and OFF-target editing efficiencies for AAVS1 and ON-target efficiencies for EMX1, RUNX1, and HEK. OFF-target editing measurements for EMX1, RUNX1, and HEK were used for model validation. ( D and E ) Kinetic insulation of ON- and OFF-target editing by Cas-Acr variants. (D) Data points are shown together with inhibitor strengths as estimated by model fitting. Kinetic insulation is achieved for inhibitor strengths that fall between sigmoidal curves for ON- and OFF-target editing. (E) The calibrated model can be used to predict the ratio between ON- and OFF-target editing efficiencies resulting from Cas-Acr variants. The inhibitor strength was defined as the fold change relative to the inhibition rate of Cas-Acr wt, i.e., Cas9 fused to wild-type AcrIIA4. Lines show model simulations; circles indicate measured data points. ( F ) Model simulations of the ratio between ON- and OFF-target editing efficiencies relative to ON-target editing efficiency illustrate the trade-off between Cas9 fidelity and ON-target editing efficiency. Cas-Acr variants can be selected on the basis of highest tolerated OFF-target editing efficiencies.
    Figure Legend Snippet: A mathematical model of Cas-Acr action explains improved specificity and informs experimental planning. ( A ) Overview of the mathematical model of gene editing with Cas-Acr constructs. The model accounts for turnover of plasmids, sgRNA, Cas-Acr mRNA, and protein; transition between the active and inhibited states (Cas-Acr inh ); sgRNA binding (Cas-Acr:sgRNA, Cas-Acr inh :sgRNA); association with a target gene; and gene editing. ( B ) Exemplary model fits to time-resolved T7 endonuclease assay measurements using the AAVS1-targeting sgRNA and either wild-type Cas9 or the Cas-Acr variant Ins. 5 (see fig. S10 for the full set of fits). ( C ) ON- and OFF-target editing efficiencies for sgRNAs targeting the AAVS1, EMX1, RUNX1, or HEK locus are shown together with model simulations of editing efficiencies for either wild-type Cas9 or the indicated Cas-Acr variants. The model was calibrated with ON- and OFF-target editing efficiencies for AAVS1 and ON-target efficiencies for EMX1, RUNX1, and HEK. OFF-target editing measurements for EMX1, RUNX1, and HEK were used for model validation. ( D and E ) Kinetic insulation of ON- and OFF-target editing by Cas-Acr variants. (D) Data points are shown together with inhibitor strengths as estimated by model fitting. Kinetic insulation is achieved for inhibitor strengths that fall between sigmoidal curves for ON- and OFF-target editing. (E) The calibrated model can be used to predict the ratio between ON- and OFF-target editing efficiencies resulting from Cas-Acr variants. The inhibitor strength was defined as the fold change relative to the inhibition rate of Cas-Acr wt, i.e., Cas9 fused to wild-type AcrIIA4. Lines show model simulations; circles indicate measured data points. ( F ) Model simulations of the ratio between ON- and OFF-target editing efficiencies relative to ON-target editing efficiency illustrate the trade-off between Cas9 fidelity and ON-target editing efficiency. Cas-Acr variants can be selected on the basis of highest tolerated OFF-target editing efficiencies.

    Techniques Used: Construct, Binding Assay, Variant Assay, Inhibition

    11) Product Images from "MYC CONTROLS THE EPSTEIN-BARR VIRUS LYTIC SWITCH"

    Article Title: MYC CONTROLS THE EPSTEIN-BARR VIRUS LYTIC SWITCH

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2020.03.025

    MYC Occupancy of EBV Genome oriLyt E-Box Sites Maintains Latency in BL (A) ChIP-seq analysis of EBV genome MYC occupancy and ATAC-seq analysis of effects of MYC depletion on EBV genomic accessibility. Shown are Daudi BL EBV genome input and MYC ChIP-seq tracks. Background-subtracted peak calling identified seven significant EBV genomic peaks, highlighted by black bars. Values at top left indicate track heights. n=2 ATAC-seq tracks from Akata with indicated sgRNAs and treated with acyclovir (100 μg/ml). Zoomed tracks of oriLyt regions are shown. Akata EBV DNA sequences of sgRNA-targeted regions are shown, with E-boxes in red. (B) T7E1 assay of Cas9 oriLyt region editing. Representative T7E1 nuclease-treated PCR products are shown. (C) ChIP-qPCR of MYC oriLyt region occupancy. Ig-control or anti-HA ChIP was performed on chromatin from Akata with HA-MYC and indicated sgRNA expression. qPCR was done with E-box regions primers. Mean + SD are shown for n=3 replicates. ****p
    Figure Legend Snippet: MYC Occupancy of EBV Genome oriLyt E-Box Sites Maintains Latency in BL (A) ChIP-seq analysis of EBV genome MYC occupancy and ATAC-seq analysis of effects of MYC depletion on EBV genomic accessibility. Shown are Daudi BL EBV genome input and MYC ChIP-seq tracks. Background-subtracted peak calling identified seven significant EBV genomic peaks, highlighted by black bars. Values at top left indicate track heights. n=2 ATAC-seq tracks from Akata with indicated sgRNAs and treated with acyclovir (100 μg/ml). Zoomed tracks of oriLyt regions are shown. Akata EBV DNA sequences of sgRNA-targeted regions are shown, with E-boxes in red. (B) T7E1 assay of Cas9 oriLyt region editing. Representative T7E1 nuclease-treated PCR products are shown. (C) ChIP-qPCR of MYC oriLyt region occupancy. Ig-control or anti-HA ChIP was performed on chromatin from Akata with HA-MYC and indicated sgRNA expression. qPCR was done with E-box regions primers. Mean + SD are shown for n=3 replicates. ****p

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing

    12) Product Images from "Genome editing via delivery of Cas9 ribonucleoprotein"

    Article Title: Genome editing via delivery of Cas9 ribonucleoprotein

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2017.04.003

    Analysis of a T7 endonuclease assay on a 10% polyacrylamide TBE-urea gel (A) and a 2% agarose gel (B). After denaturation and annealing, pairs of samples were run without (−) or with (+) T7 endonuclease (T7E1) treatment. The numbers indicate the identities of the sgRNAs; marker lanes are unlabeled.
    Figure Legend Snippet: Analysis of a T7 endonuclease assay on a 10% polyacrylamide TBE-urea gel (A) and a 2% agarose gel (B). After denaturation and annealing, pairs of samples were run without (−) or with (+) T7 endonuclease (T7E1) treatment. The numbers indicate the identities of the sgRNAs; marker lanes are unlabeled.

    Techniques Used: Agarose Gel Electrophoresis, Marker

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    Agarose Gel Electrophoresis:

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    New England Biolabs t7 endonuclease i
    Deletion of MCM10 through CRISPR/Cas9 technology. Notes:  ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P
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    Deletion of MCM10 through CRISPR/Cas9 technology. Notes:  ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: Ablation of MCM10 using CRISPR/Cas9 restrains the growth and migration of esophageal squamous cell carcinoma cells through inhibition of Akt signaling

    doi: 10.2147/OTT.S157025

    Figure Lengend Snippet: Deletion of MCM10 through CRISPR/Cas9 technology. Notes: ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P

    Article Snippet: After treatment with T7 endonuclease I (New England Biolabs, Ipswich, MA, USA) at 37°C for 2 h, the resulting fragments were subjected to 1% agarose gel electrophoresis and stained with ethidium bromide.

    Techniques: CRISPR, Knock-Out, Construct, Transfection, Fluorescence, Sequencing, Clone Assay, Derivative Assay, Polymerase Chain Reaction, Amplification, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Migration, In Vitro, Wound Healing Assay

    Detection of CRISPR/Cas9-induced indel mutations in the murine  Ramp2  gene at the single blastocyst level. A : Schema of the  Ramp2  guide RNA (R2gRNA) targeting exon 1 in the murine  Ramp2  gene. The R2gRNA-coding sequence is shown in blue. The protospacer-adjacent motif (PAM) sequence is shown in red. The arrows indicate the locations of the PCR primers (see Table   3 ).  B : Agarose gel electrophoresis of T7 endonuclease I-treated PCR products and surveyor-treated PCR products derived from 8 individual blastocysts (see Table   1 , experiment 3). Lanes 1–8: PCR products amplified from the crude DNA solution of each blastocyst. Lane NIC (no injection control): PCR product amplified from the crude DNA of a single uninjected control blastocyst. M: lambda  Hind III + 100-bp ladder markers.  C : Sequencing results for the PCR products shown in lanes 1–8 and NIC of (B). The result of each sample was summarized as the two Ramp2 alleles. “wt” indicates the wild-type sequence. “-” indicates base deletion.  D : Gel electrophoretic pattern of DNA products after whole genome amplification (WGA) of the crude DNA shown in lanes 1–8 and NIC of (B) (top panel) and after subsequent PCR amplification (middle panel) and T7 endonuclease I digestion (bottom panel). Note that the band pattern is similar to that shown in (B). NTC (no template control) indicates the WGA product obtained using water in place of template as a negative control (top panel); no PCR products were amplified by PCR (middle panel).

    Journal: BMC Biotechnology

    Article Title: A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice

    doi: 10.1186/1472-6750-14-69

    Figure Lengend Snippet: Detection of CRISPR/Cas9-induced indel mutations in the murine Ramp2 gene at the single blastocyst level. A : Schema of the Ramp2 guide RNA (R2gRNA) targeting exon 1 in the murine Ramp2 gene. The R2gRNA-coding sequence is shown in blue. The protospacer-adjacent motif (PAM) sequence is shown in red. The arrows indicate the locations of the PCR primers (see Table  3 ). B : Agarose gel electrophoresis of T7 endonuclease I-treated PCR products and surveyor-treated PCR products derived from 8 individual blastocysts (see Table  1 , experiment 3). Lanes 1–8: PCR products amplified from the crude DNA solution of each blastocyst. Lane NIC (no injection control): PCR product amplified from the crude DNA of a single uninjected control blastocyst. M: lambda Hind III + 100-bp ladder markers. C : Sequencing results for the PCR products shown in lanes 1–8 and NIC of (B). The result of each sample was summarized as the two Ramp2 alleles. “wt” indicates the wild-type sequence. “-” indicates base deletion. D : Gel electrophoretic pattern of DNA products after whole genome amplification (WGA) of the crude DNA shown in lanes 1–8 and NIC of (B) (top panel) and after subsequent PCR amplification (middle panel) and T7 endonuclease I digestion (bottom panel). Note that the band pattern is similar to that shown in (B). NTC (no template control) indicates the WGA product obtained using water in place of template as a negative control (top panel); no PCR products were amplified by PCR (middle panel).

    Article Snippet: Mutational assays with T7 endonuclease I and surveyor nuclease and direct sequencing CRISPR/Cas9 -induced indel mutations were detected through mutational assays with T7 endonuclease I and Surveyor nuclease and through direct sequencing of the PCR products.

    Techniques: CRISPR, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Derivative Assay, Amplification, Injection, Whole Genome Amplification, Negative Control

    Comparison of assays using Surveyor nuclease and T7 endonuclease I. A : P1 and P2 polymerase chain reaction (PCR) products (approximately 800 bp) were used as template to evaluate which nuclease provide clearer and more reproducible results. The P1 and P2 PCR products were generated by PCR of pC2EpA or pCEpA, respectively, using the 3525A and bGpA2 primer set. Both 800-bp products re-annealed were expected to cleavage into two fragments (525 and 275 bp in size) by Surveyor nuclease or T7 endonuclease I.  B : Agarose gel electrophoresis of nuclease-treated PCR products. A mixture containing P1 (100 ng) and P1 (100 ng) (lane 1), P2 (100 ng) and P2 (100 ng) (lane 2), or P1 (100 ng) and P2 (100 ng) (lane 3) was re-annealed and treated with each nuclease. In addition, a mixture containing P1 (100 ng) and P2 (100 ng) was directly treated with nuclease without re-annealing (lane 4). The 2 asterisks indicate the expected 525- and 275-bp cleavage bands. The experiments were carried out 3 times. The representative results were shown here.

    Journal: BMC Biotechnology

    Article Title: A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice

    doi: 10.1186/1472-6750-14-69

    Figure Lengend Snippet: Comparison of assays using Surveyor nuclease and T7 endonuclease I. A : P1 and P2 polymerase chain reaction (PCR) products (approximately 800 bp) were used as template to evaluate which nuclease provide clearer and more reproducible results. The P1 and P2 PCR products were generated by PCR of pC2EpA or pCEpA, respectively, using the 3525A and bGpA2 primer set. Both 800-bp products re-annealed were expected to cleavage into two fragments (525 and 275 bp in size) by Surveyor nuclease or T7 endonuclease I. B : Agarose gel electrophoresis of nuclease-treated PCR products. A mixture containing P1 (100 ng) and P1 (100 ng) (lane 1), P2 (100 ng) and P2 (100 ng) (lane 2), or P1 (100 ng) and P2 (100 ng) (lane 3) was re-annealed and treated with each nuclease. In addition, a mixture containing P1 (100 ng) and P2 (100 ng) was directly treated with nuclease without re-annealing (lane 4). The 2 asterisks indicate the expected 525- and 275-bp cleavage bands. The experiments were carried out 3 times. The representative results were shown here.

    Article Snippet: Mutational assays with T7 endonuclease I and surveyor nuclease and direct sequencing CRISPR/Cas9 -induced indel mutations were detected through mutational assays with T7 endonuclease I and Surveyor nuclease and through direct sequencing of the PCR products.

    Techniques: Polymerase Chain Reaction, Generated, Agarose Gel Electrophoresis

    Detection of CRISPR/Cas9-induced mutations at the  mCherry  locus in  mylz2-mCherry  transgenic amphioxus. ( A ) T7EI cleavage assay showing no detectable mutation in uninjected embryos (control), or embryos injected with  mCherry -sgRNA and  Cas9  mRNA transcribed from pXT7-Cas9 (pXT7) or pCS2-nls-zCas9-nls (pCS2) plasmids. —means no T7 endonuclease I was added and + means T7 endonuclease I was added. ( B ) RT-qPCR analysis of  Cas9  mRNA and  mCherry -sgRNA expression in embryos injected with Cas9 protein and  mCherry -sgRNA or  Cas9  mRNA and  mCherry -sgRNA. Samples were collected and examined at unfertilized egg (egg), two-cell (2-cell) and early gastrula (EG) stages. ( C ) T7EI cleavage assay showing no detectable mutation in uninjected embryos (control) and different levels of mutations in embryos injected, respectively, with  mCherry -sgRNA and each of the three commercial Cas9 protein (TaKaRa, Thermo and NEB). —means no T7 endonuclease I was added and + means T7 endonuclease I was added. ( D ) Mutations were detected using DNA sequencing in the injected embryos. The wild-type (WT) reference sequence is shown on the top. The underlined sequence is the target site and GGG (green) is the PAM sequence. Deletion is shown by the dashed line and insertion is highlighted by inserted letters. Indels (+, insertion; –, deletion) are listed on the right of each allele. ( E ) Mutation efficiencies in embryos injected with different molar ratios of TaKaRa Cas9 to  mCherry -sgRNA in which the amount of Cas9 protein was kept constant while that of  mCherry -sgRNA was different. ( F ) Mutation efficiencies in embryos injected with different molar ratios of TaKaRa Cas9 to  mCherry -sgRNA in which amount of  mCherry -sgRNA was kept constant while that of Cas9 protein was different. Red arrowheads in panels C, E, F, H and I mark bands released by T7 endonuclease I digestion. ( G ) Observation of red fluorescent signal in two-day transgenetic larvae. In contrast to uninjected (control) larvae which showed a specific fluorescent signal in the notochord and somites, the injected embryos showed no detectable fluorescent signals. The blue fluorescent signal shows the amphioxus cell nuclei stained with DAPI. Scale bars in all images are 100 μm. ( H , I ) Mutation efficiencies in different stages of embryos injected with TaKaRa Cas9 and  mCherry -sgRNA. Un-eggs, unfertilized eggs; LB, late blastula; MG, mid-gastrula; RS, rotation stage; LM, late neurula; MO, mouth-opening stage.

    Journal: Genes

    Article Title: Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

    doi: 10.3390/genes11111311

    Figure Lengend Snippet: Detection of CRISPR/Cas9-induced mutations at the mCherry locus in mylz2-mCherry transgenic amphioxus. ( A ) T7EI cleavage assay showing no detectable mutation in uninjected embryos (control), or embryos injected with mCherry -sgRNA and Cas9 mRNA transcribed from pXT7-Cas9 (pXT7) or pCS2-nls-zCas9-nls (pCS2) plasmids. —means no T7 endonuclease I was added and + means T7 endonuclease I was added. ( B ) RT-qPCR analysis of Cas9 mRNA and mCherry -sgRNA expression in embryos injected with Cas9 protein and mCherry -sgRNA or Cas9 mRNA and mCherry -sgRNA. Samples were collected and examined at unfertilized egg (egg), two-cell (2-cell) and early gastrula (EG) stages. ( C ) T7EI cleavage assay showing no detectable mutation in uninjected embryos (control) and different levels of mutations in embryos injected, respectively, with mCherry -sgRNA and each of the three commercial Cas9 protein (TaKaRa, Thermo and NEB). —means no T7 endonuclease I was added and + means T7 endonuclease I was added. ( D ) Mutations were detected using DNA sequencing in the injected embryos. The wild-type (WT) reference sequence is shown on the top. The underlined sequence is the target site and GGG (green) is the PAM sequence. Deletion is shown by the dashed line and insertion is highlighted by inserted letters. Indels (+, insertion; –, deletion) are listed on the right of each allele. ( E ) Mutation efficiencies in embryos injected with different molar ratios of TaKaRa Cas9 to mCherry -sgRNA in which the amount of Cas9 protein was kept constant while that of mCherry -sgRNA was different. ( F ) Mutation efficiencies in embryos injected with different molar ratios of TaKaRa Cas9 to mCherry -sgRNA in which amount of mCherry -sgRNA was kept constant while that of Cas9 protein was different. Red arrowheads in panels C, E, F, H and I mark bands released by T7 endonuclease I digestion. ( G ) Observation of red fluorescent signal in two-day transgenetic larvae. In contrast to uninjected (control) larvae which showed a specific fluorescent signal in the notochord and somites, the injected embryos showed no detectable fluorescent signals. The blue fluorescent signal shows the amphioxus cell nuclei stained with DAPI. Scale bars in all images are 100 μm. ( H , I ) Mutation efficiencies in different stages of embryos injected with TaKaRa Cas9 and mCherry -sgRNA. Un-eggs, unfertilized eggs; LB, late blastula; MG, mid-gastrula; RS, rotation stage; LM, late neurula; MO, mouth-opening stage.

    Article Snippet: In the T7EI cleavage assay, 20–40 ng amplicons were melted and reannealed in a 10 μL reaction system as follows: 95 °C for 5 min, 95 °C to 20 °C ramping at 0.1 °C/s and holding at 20 °C, then digested by 2U of T7 endonuclease I (T7E1, New England BioLabs, Ipswich, MA, USA) at 37 °C for 15 min; in the restriction enzyme digestion assay, 20–40 ng amplicons were digested in a 10 μL reaction system with the proper enzymes.

    Techniques: CRISPR, Transgenic Assay, Cleavage Assay, Mutagenesis, Injection, Quantitative RT-PCR, Expressing, DNA Sequencing, Sequencing, Staining

    Generation of RHD -mutated erythroid progenitor cells. ( a ) Schematic representation illustrating the process of RHD -mutated clone generation. Clonal culture of HiDEP-1 erythroid progenitor cells was initiated 3 days after transfection with plasmids encoding TALENs that target RHD . Genomic DNA from each clone was analysed 17 days after the initiation of clonal culture. ( b ) T7E1-based clonal analysis. The genomic DNA isolated from each clone was subjected to the T7E1 assay. Arrows indicate the expected position of DNA bands cleaved by T7E1. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Clones containing mutations in the target sites were marked with red clone numbers. Untransfected cells and a cell population transfected with the TALEN plasmids were used as the negative control (NC) and positive control (PC), respectively. M: Markers

    Journal: Nature Communications

    Article Title: Rh D blood group conversion using transcription activator-like effector nucleases

    doi: 10.1038/ncomms8451

    Figure Lengend Snippet: Generation of RHD -mutated erythroid progenitor cells. ( a ) Schematic representation illustrating the process of RHD -mutated clone generation. Clonal culture of HiDEP-1 erythroid progenitor cells was initiated 3 days after transfection with plasmids encoding TALENs that target RHD . Genomic DNA from each clone was analysed 17 days after the initiation of clonal culture. ( b ) T7E1-based clonal analysis. The genomic DNA isolated from each clone was subjected to the T7E1 assay. Arrows indicate the expected position of DNA bands cleaved by T7E1. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Clones containing mutations in the target sites were marked with red clone numbers. Untransfected cells and a cell population transfected with the TALEN plasmids were used as the negative control (NC) and positive control (PC), respectively. M: Markers

    Article Snippet: The PCR amplicons were denatured by heating, annealed to form heteroduplex DNA, treated with 5 units of mismatch-sensitive T7 endonuclease 1 (New England Biolabs, Hitchin, UK) for 20 min at 37 °C and analysed by 2% agarose gel electrophoresis.

    Techniques: Transfection, TALENs, Isolation, Marker, Clone Assay, Negative Control, Positive Control

    TALENs targeting the human RHD gene. ( a ) Schematic of the TALEN-targeting sites in the RHD gene. Blue boxes indicate exons. RHD _E1_TALENs and RHD _E4_TALENs represent the TALEN pairs that target sequences (shown in a red colour) in exon 1 and exon 4, respectively. The red, yellow, green and purple rectangular boxes in the TALENs symbolize the TALE repeat units that recognize guanine, thymine, cytosine and adenine, respectively. ( b , c ) T7E1 assay using 293T cells after transfection with plasmids encoding TALENs targeting RHD exon 1 ( b , RHD _E1_TALENs) or exon 4 ( c , RHD _E4_TALENs), respectively. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Arrows indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gel indicate mutation percentages measured by band intensities.

    Journal: Nature Communications

    Article Title: Rh D blood group conversion using transcription activator-like effector nucleases

    doi: 10.1038/ncomms8451

    Figure Lengend Snippet: TALENs targeting the human RHD gene. ( a ) Schematic of the TALEN-targeting sites in the RHD gene. Blue boxes indicate exons. RHD _E1_TALENs and RHD _E4_TALENs represent the TALEN pairs that target sequences (shown in a red colour) in exon 1 and exon 4, respectively. The red, yellow, green and purple rectangular boxes in the TALENs symbolize the TALE repeat units that recognize guanine, thymine, cytosine and adenine, respectively. ( b , c ) T7E1 assay using 293T cells after transfection with plasmids encoding TALENs targeting RHD exon 1 ( b , RHD _E1_TALENs) or exon 4 ( c , RHD _E4_TALENs), respectively. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Arrows indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gel indicate mutation percentages measured by band intensities.

    Article Snippet: The PCR amplicons were denatured by heating, annealed to form heteroduplex DNA, treated with 5 units of mismatch-sensitive T7 endonuclease 1 (New England Biolabs, Hitchin, UK) for 20 min at 37 °C and analysed by 2% agarose gel electrophoresis.

    Techniques: TALENs, Transfection, Marker, Mutagenesis