cas9 enzyme  (New England Biolabs)


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  • 99
    Name:
    Cas9 Nuclease S pyogenes
    Description:

    Catalog Number:
    M0386
    Price:
    540
    Category:
    DNA Modifying Enzymes
    Applications:
    Functional Genomics
    Size:
    2000 pmol
    Buy from Supplier


    Structured Review

    New England Biolabs cas9 enzyme
    Cas9 Nuclease S pyogenes

    https://www.bioz.com/result/cas9 enzyme/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas9 enzyme - by Bioz Stars, 2021-08
    99/100 stars

    Images

    1) Product Images from "CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool"

    Article Title: CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124633

    Experimental verification of sgRNA-1. (A) In vitro cleavage depending on sgRNA-1/Cas9 occurred on linearized plasmids containing eGFP but not eGFP var . Successful cleavage of eGFP plasmid (4128bp) resulted in a 2052bp and 2076bp fragment. The absence of expected fragments (627bp, 4025bp) demonstrated that eGFP var (4652bp) was not digested by sgRNA-1/Cas9. (B) A faint double band (2154bp, 2198bp, asterisk) indicated inefficient digestion of off-target 1 (OT#1) while OT#2 and OT#3 ( S1 Table ) were not cleaved. Note: contrast was enhanced for better visualization. (C) Silent mutations of sgRNA-1 target site in eGFP var . (D-E) Injections of sgRNA-1 and Cas9 mRNA into wimb -/+ embryos (D) resulted in strong inactivation of eGFP (E).
    Figure Legend Snippet: Experimental verification of sgRNA-1. (A) In vitro cleavage depending on sgRNA-1/Cas9 occurred on linearized plasmids containing eGFP but not eGFP var . Successful cleavage of eGFP plasmid (4128bp) resulted in a 2052bp and 2076bp fragment. The absence of expected fragments (627bp, 4025bp) demonstrated that eGFP var (4652bp) was not digested by sgRNA-1/Cas9. (B) A faint double band (2154bp, 2198bp, asterisk) indicated inefficient digestion of off-target 1 (OT#1) while OT#2 and OT#3 ( S1 Table ) were not cleaved. Note: contrast was enhanced for better visualization. (C) Silent mutations of sgRNA-1 target site in eGFP var . (D-E) Injections of sgRNA-1 and Cas9 mRNA into wimb -/+ embryos (D) resulted in strong inactivation of eGFP (E).

    Techniques Used: In Vitro, Plasmid Preparation

    2) Product Images from "CaBagE: A Cas9-based Back ground Elimination strategy for targeted, long-read DNA sequencing"

    Article Title: CaBagE: A Cas9-based Back ground Elimination strategy for targeted, long-read DNA sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0241253

    Schematic of Cas9 background elimination strategy. A) Cas9 is bound to either side of target sequence. B) Off-target DNA is digested with a combination of exonucleases. C) Heat is used to dissociate that Cas9 and inactivate the exonucleases. D) On-target fragment is available for A-tailing and sequence adapter ligation. E) Target fragments are sequenced on the MinlON for 48 hours.
    Figure Legend Snippet: Schematic of Cas9 background elimination strategy. A) Cas9 is bound to either side of target sequence. B) Off-target DNA is digested with a combination of exonucleases. C) Heat is used to dissociate that Cas9 and inactivate the exonucleases. D) On-target fragment is available for A-tailing and sequence adapter ligation. E) Target fragments are sequenced on the MinlON for 48 hours.

    Techniques Used: Sequencing, Ligation

    A. gBlock assay design for Cas9 challenge with exonuclease. gBlock contained two pairs of gRNA target sites, one with PAM-out orientation and one with PAM-in orientation. Upon Cas9 binding (depicted by scissors), each set of target sites generate 3 unique fragment lengths. The gRNAs are represented as dotted lines. B. Capillary electrophoresis results from exonuclease challenge experiment with Cas9. 15nM gBlock DNA was incubated with 40nM ribonucleoprotien complex, followed by digestion with a combination of exonucleases for 2 hours. When Cas9 is used without exonucleases, the gBlock is cut to produce expected fragment lengths. Upon challenge with exonuclease, only the fragments flanked on both sides by Cas9 remain in the sample. (l = in; O = out).
    Figure Legend Snippet: A. gBlock assay design for Cas9 challenge with exonuclease. gBlock contained two pairs of gRNA target sites, one with PAM-out orientation and one with PAM-in orientation. Upon Cas9 binding (depicted by scissors), each set of target sites generate 3 unique fragment lengths. The gRNAs are represented as dotted lines. B. Capillary electrophoresis results from exonuclease challenge experiment with Cas9. 15nM gBlock DNA was incubated with 40nM ribonucleoprotien complex, followed by digestion with a combination of exonucleases for 2 hours. When Cas9 is used without exonucleases, the gBlock is cut to produce expected fragment lengths. Upon challenge with exonuclease, only the fragments flanked on both sides by Cas9 remain in the sample. (l = in; O = out).

    Techniques Used: Binding Assay, Electrophoresis, Incubation

    3) Product Images from "Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos"

    Article Title: Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345256

    Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange
    Figure Legend Snippet: Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange

    Techniques Used: Injection

    4) Product Images from "Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge *"

    Article Title: Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.C116.758706

    Time course of Cas9 cleavage on naked DNA and nucleosome substrates containing sgRNA-target DNA mismatches. A , diagram showing the location of mismatches within the sgRNA relative to the DNA target ( white arrow ) in the 289-bp substrate containing the 601 nucleosome positioning sequence. The break in the arrow denotes the Cas9 cleavage site, and the point of the arrow corresponds to the PAM site. The locations and sequences of the sgRNA mismatches are indicated, with the mismatch location ( e.g. +10) denoting nucleotide distance from the PAM. B , representative polyacrylamide gels showing the full-length substrate and Cas9 cleavage product of the naked DNA substrate for each sgRNA at different time points. No Cas9 represents control samples with only the wt-sgRNA. C , graph showing the percentage of cleavage of the naked DNA substrate as a function of time. Data points represent the average of three independent experiments, and error bars represent standard deviations. D , same as in panel B , except showing the time course of Cas9 cleavage of nucleosome substrate. E , summary of Cas9 cleavage of nucleosome substrates for the indicated sgRNAs. Data points represent the average of three independent experiments, and error bars represent standard deviations.
    Figure Legend Snippet: Time course of Cas9 cleavage on naked DNA and nucleosome substrates containing sgRNA-target DNA mismatches. A , diagram showing the location of mismatches within the sgRNA relative to the DNA target ( white arrow ) in the 289-bp substrate containing the 601 nucleosome positioning sequence. The break in the arrow denotes the Cas9 cleavage site, and the point of the arrow corresponds to the PAM site. The locations and sequences of the sgRNA mismatches are indicated, with the mismatch location ( e.g. +10) denoting nucleotide distance from the PAM. B , representative polyacrylamide gels showing the full-length substrate and Cas9 cleavage product of the naked DNA substrate for each sgRNA at different time points. No Cas9 represents control samples with only the wt-sgRNA. C , graph showing the percentage of cleavage of the naked DNA substrate as a function of time. Data points represent the average of three independent experiments, and error bars represent standard deviations. D , same as in panel B , except showing the time course of Cas9 cleavage of nucleosome substrate. E , summary of Cas9 cleavage of nucleosome substrates for the indicated sgRNAs. Data points represent the average of three independent experiments, and error bars represent standard deviations.

    Techniques Used: Sequencing

    Model of how nucleosomes impact Cas9 activity at off-target sites containing mismatches. A , Cas9 cannot efficiently cleave target sites in strongly positioned nucleosomes if the PAM site ( yellow rectangle ) is located within the nucleosome. B , Cas9 can efficiently cleave target sites in the nucleosome if the PAM site ( yellow rectangle ) is located in the accessible linker DNA. C , a single mismatch between the sgRNA and DNA target ( red asterisk ) inhibits Cas9 cleavage of nucleosome substrates, particularly if the mismatch occurs in the PAM-proximal seed region. D , Cas9 can efficiently cleave naked DNA substrates containing single mismatches ( red asterisk ).
    Figure Legend Snippet: Model of how nucleosomes impact Cas9 activity at off-target sites containing mismatches. A , Cas9 cannot efficiently cleave target sites in strongly positioned nucleosomes if the PAM site ( yellow rectangle ) is located within the nucleosome. B , Cas9 can efficiently cleave target sites in the nucleosome if the PAM site ( yellow rectangle ) is located in the accessible linker DNA. C , a single mismatch between the sgRNA and DNA target ( red asterisk ) inhibits Cas9 cleavage of nucleosome substrates, particularly if the mismatch occurs in the PAM-proximal seed region. D , Cas9 can efficiently cleave naked DNA substrates containing single mismatches ( red asterisk ).

    Techniques Used: Activity Assay

    Cas9 activity with a mismatch sgRNA is restored with complementary mutation in 601 nucleosome substrate. A , diagram showing mismatch sgRNAs and complementary mutation in the 601 nucleosome substrate at position +6 nucleotides from the PAM site. B , representative reconstitutions of the wild-type 601 sequence and mutant DNA substrate (601-m6, with sequence change to complement the sgRNA +6 mismatch) into mononucleosomes analyzed by native polyacrylamide gel electrophoresis. NCP indicates the band corresponding to reconstituted nucleosome core particles. C , representative polyacrylamide gels showing cleavage of the 601-m6 naked DNA ( top panel ) and nucleosome ( bottom panel ) substrates after 30 min by Cas9 targeted by different sgRNAs used in the study. No sgRNA represents control samples with only the Cas9 enzyme (1 pmol) without the addition of an sgRNA. D , graph showing the percentage of cleavage of the 601-m6 DNA and nucleosome substrates by Cas9. Note that the +2, +10, and +18 sgRNAs have two mismatches with the 601-m6 substrate, because all have the equivalent of an additional +6 mismatch. Data points represent the average of at least three independent experiments, and error bars represent standard deviations.
    Figure Legend Snippet: Cas9 activity with a mismatch sgRNA is restored with complementary mutation in 601 nucleosome substrate. A , diagram showing mismatch sgRNAs and complementary mutation in the 601 nucleosome substrate at position +6 nucleotides from the PAM site. B , representative reconstitutions of the wild-type 601 sequence and mutant DNA substrate (601-m6, with sequence change to complement the sgRNA +6 mismatch) into mononucleosomes analyzed by native polyacrylamide gel electrophoresis. NCP indicates the band corresponding to reconstituted nucleosome core particles. C , representative polyacrylamide gels showing cleavage of the 601-m6 naked DNA ( top panel ) and nucleosome ( bottom panel ) substrates after 30 min by Cas9 targeted by different sgRNAs used in the study. No sgRNA represents control samples with only the Cas9 enzyme (1 pmol) without the addition of an sgRNA. D , graph showing the percentage of cleavage of the 601-m6 DNA and nucleosome substrates by Cas9. Note that the +2, +10, and +18 sgRNAs have two mismatches with the 601-m6 substrate, because all have the equivalent of an additional +6 mismatch. Data points represent the average of at least three independent experiments, and error bars represent standard deviations.

    Techniques Used: Activity Assay, Mutagenesis, Sequencing, Polyacrylamide Gel Electrophoresis

    5) Product Images from "GFAT2 and AMDHD2 act in tandem to control the hexosamine biosynthetic pathway"

    Article Title: GFAT2 and AMDHD2 act in tandem to control the hexosamine biosynthetic pathway

    Journal: bioRxiv

    doi: 10.1101/2021.04.23.441115

    Chemical mutagenesis screen for tunicamycin resistance in mESCs identifies AMDHD2 (A) Schematic representation of experimental workflow for TM resistance screen using ENU mutagenesis in combination with whole exome sequencing. (B) Schematic representation of the mouse Amdhd2 locus. Amino acid substitutions identified in the screen are highlighted. (C) Western blot analysis of CRISPR/Cas9 generated AMDHD2 K.O. AN3-12 mESCs compared to wildtype cells (ctrl). (D) Cell viability (XTT assay) of WT and AMDHD2 K.O. AN3-12 cells treated with 0.5 µg/ml TM for 48h (mean ± SEM, n=3, ** p
    Figure Legend Snippet: Chemical mutagenesis screen for tunicamycin resistance in mESCs identifies AMDHD2 (A) Schematic representation of experimental workflow for TM resistance screen using ENU mutagenesis in combination with whole exome sequencing. (B) Schematic representation of the mouse Amdhd2 locus. Amino acid substitutions identified in the screen are highlighted. (C) Western blot analysis of CRISPR/Cas9 generated AMDHD2 K.O. AN3-12 mESCs compared to wildtype cells (ctrl). (D) Cell viability (XTT assay) of WT and AMDHD2 K.O. AN3-12 cells treated with 0.5 µg/ml TM for 48h (mean ± SEM, n=3, ** p

    Techniques Used: Mutagenesis, Sequencing, Western Blot, CRISPR, Generated, XTT Assay

    Structural and biochemical characterization of human AMDHD2 Generation of different AMDHD2 K.O. founder mice. (A) Schematic of the CRISPR/Cas9 targeted exon of the mouse Amdhd2 locus. Deletions in founder lines 1-4 are indicated in red. (B) Table listing used guide combinations and deletion details of the AMDHD2 K.O. founder lines. (C) Representative genotyping results of AMDHD2 K.O. mice. The WT PCR product is 675 bp and the Amdhd2 K.O. allele shows a size of 300 bp (line 902).
    Figure Legend Snippet: Structural and biochemical characterization of human AMDHD2 Generation of different AMDHD2 K.O. founder mice. (A) Schematic of the CRISPR/Cas9 targeted exon of the mouse Amdhd2 locus. Deletions in founder lines 1-4 are indicated in red. (B) Table listing used guide combinations and deletion details of the AMDHD2 K.O. founder lines. (C) Representative genotyping results of AMDHD2 K.O. mice. The WT PCR product is 675 bp and the Amdhd2 K.O. allele shows a size of 300 bp (line 902).

    Techniques Used: Mouse Assay, CRISPR, Polymerase Chain Reaction

    6) Product Images from "Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH)"

    Article Title: Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH)

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky411

    Schematic representation of the CATCH method. Peripheral blood mononuclear cells were embedded in an agarose gel-plug and lysed. Genomic DNA was cleaved in the plug using guided Cas9, and the target DNA was separated by PFGE. The desired band (indicated by an arrow) was excised from the gel, and the DNA was isolated, purified and analyzed.
    Figure Legend Snippet: Schematic representation of the CATCH method. Peripheral blood mononuclear cells were embedded in an agarose gel-plug and lysed. Genomic DNA was cleaved in the plug using guided Cas9, and the target DNA was separated by PFGE. The desired band (indicated by an arrow) was excised from the gel, and the DNA was isolated, purified and analyzed.

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Purification

    7) Product Images from "Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos"

    Article Title: Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345256

    Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange
    Figure Legend Snippet: Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange

    Techniques Used: Injection

    8) Product Images from "CRISPR/Cas9 mediated mutations as a new tool for studying taste in honeybees"

    Article Title: CRISPR/Cas9 mediated mutations as a new tool for studying taste in honeybees

    Journal: bioRxiv

    doi: 10.1101/2020.03.26.009696

    CRISPR/Cas9 induced nucleotide changes at the AmGr3 gene target the second putative transmembrane domain and introduce double-nonsense mutations (ns/ns) at high frequency. The graph shows that the mRNA target-site for AmGr3 (5’-gcaacttgtagtgatgtgcttgg-3’) is placed within the putative second transmembrane domain (TMD) after the N-terminus. Folding predictions (I-TASSER, PHYRE-Protein and TMHMM) show different outcomes about a possible upstream TMD (TMD 0). The two possible frameshift mutations (not a multiple of three) driven from the sgRNA target-site introduce either a stop codon at position 103 aa or 129 aa (amino acids) of the deduced sequence and are followed by multiple stops. As a consequence, five TMDs of the AmGr3 proteins are lacking in double-nonsense (ns/ns) mutants so it is assumed not to function as a fructose receptor at all.
    Figure Legend Snippet: CRISPR/Cas9 induced nucleotide changes at the AmGr3 gene target the second putative transmembrane domain and introduce double-nonsense mutations (ns/ns) at high frequency. The graph shows that the mRNA target-site for AmGr3 (5’-gcaacttgtagtgatgtgcttgg-3’) is placed within the putative second transmembrane domain (TMD) after the N-terminus. Folding predictions (I-TASSER, PHYRE-Protein and TMHMM) show different outcomes about a possible upstream TMD (TMD 0). The two possible frameshift mutations (not a multiple of three) driven from the sgRNA target-site introduce either a stop codon at position 103 aa or 129 aa (amino acids) of the deduced sequence and are followed by multiple stops. As a consequence, five TMDs of the AmGr3 proteins are lacking in double-nonsense (ns/ns) mutants so it is assumed not to function as a fructose receptor at all.

    Techniques Used: CRISPR, Introduce, Sequencing

    9) Product Images from "Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos"

    Article Title: Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345256

    Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange
    Figure Legend Snippet: Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange

    Techniques Used: Injection

    10) Product Images from "Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9"

    Article Title: Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02547-w

    Modifications of the anti-mt CRISPR treatment. Compared to the treatment shown in Fig. 1 (100X gRNA, 100X Cas9, 1 h incubation), labeled “standard”, modifications in the treatment did not show improvement. The number of peaks is comparable or even lower in the modified treatments, compared to the standard treatment. Due to the low number of reads in 6 samples, the results presented were obtained with 9.8 M reads randomly sampled. See also Supplemental Fig. S3 .
    Figure Legend Snippet: Modifications of the anti-mt CRISPR treatment. Compared to the treatment shown in Fig. 1 (100X gRNA, 100X Cas9, 1 h incubation), labeled “standard”, modifications in the treatment did not show improvement. The number of peaks is comparable or even lower in the modified treatments, compared to the standard treatment. Due to the low number of reads in 6 samples, the results presented were obtained with 9.8 M reads randomly sampled. See also Supplemental Fig. S3 .

    Techniques Used: CRISPR, Incubation, Labeling, Modification

    11) Product Images from "Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9"

    Article Title: Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02547-w

    Modifications of the anti-mt CRISPR treatment. Compared to the treatment shown in Fig. 1 (100X gRNA, 100X Cas9, 1 h incubation), labeled “standard”, modifications in the treatment did not show improvement. The number of peaks is comparable or even lower in the modified treatments, compared to the standard treatment. Due to the low number of reads in 6 samples, the results presented were obtained with 9.8 M reads randomly sampled. See also Supplemental Fig. S3 .
    Figure Legend Snippet: Modifications of the anti-mt CRISPR treatment. Compared to the treatment shown in Fig. 1 (100X gRNA, 100X Cas9, 1 h incubation), labeled “standard”, modifications in the treatment did not show improvement. The number of peaks is comparable or even lower in the modified treatments, compared to the standard treatment. Due to the low number of reads in 6 samples, the results presented were obtained with 9.8 M reads randomly sampled. See also Supplemental Fig. S3 .

    Techniques Used: CRISPR, Incubation, Labeling, Modification

    12) Product Images from "Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes"

    Article Title: Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz661

    CATCH targeting and linked read sequencing of HMW DNA. ( A ) Overview of the process is illustrated. First, guide RNAs target and cut multiple genomic regions of interest. Second, target HMW DNA within the specific size range is isolated by an electrophoresis-based process. At last, the target DNA is used for linked read library preparation and sequencing. The alignment of barcode linked reads shows how sequence coverage is increased across the target segment. In the alignment plot, the X -axis indicates the reference coordinates and the Y -axis shows different barcodes representing individual HMW molecules. Dashed vertical lines indicate Cas9-gRNA cut sites. ( B ) Sequencing coverage for the target regions is shown for the three assays. For Assays 1 and 2, BRCA1 -R2 and MHC-30 libraries are shown. For Assay 3, an example of a homozygous deletion (SV1) is shown. Black bars indicate the target regions. Blue and green areas in plots indicate coverage for forward and reverse reads, respectively.
    Figure Legend Snippet: CATCH targeting and linked read sequencing of HMW DNA. ( A ) Overview of the process is illustrated. First, guide RNAs target and cut multiple genomic regions of interest. Second, target HMW DNA within the specific size range is isolated by an electrophoresis-based process. At last, the target DNA is used for linked read library preparation and sequencing. The alignment of barcode linked reads shows how sequence coverage is increased across the target segment. In the alignment plot, the X -axis indicates the reference coordinates and the Y -axis shows different barcodes representing individual HMW molecules. Dashed vertical lines indicate Cas9-gRNA cut sites. ( B ) Sequencing coverage for the target regions is shown for the three assays. For Assays 1 and 2, BRCA1 -R2 and MHC-30 libraries are shown. For Assay 3, an example of a homozygous deletion (SV1) is shown. Black bars indicate the target regions. Blue and green areas in plots indicate coverage for forward and reverse reads, respectively.

    Techniques Used: Sequencing, Isolation, Electrophoresis

    Assembly results from multiplex SV assay. ( A ) CRISPR-linked read assembly for SV1 in Assay 3 was aligned to the reference genome and compared with other long read assemblies. Red, green and blue bars indicate the portion of the assembly that aligns to the reference while a gray gap indicates no alignment. Although the gray regions have some similarity to the reference, they generally have too many homopolymer errors to successfully align. Fraction of aligned bases in the assembly is indicated at the end of the bars. ( B ) Two different sets of deletion breakpoints were determined for SV5. CRISPR-linked read SV assay captured the two SV alleles with different deletion sizes. For (A and B), the X - and Y -axes indicate the reference coordinates and the alignment of barcoded linked reads, respectively. Dashed vertical lines indicate Cas9-gRNA cut sites. ( C ) Illustration of how the breakpoints are determined in segmental duplications. Duplicated copies from GRCh38 reference genome were aligned to CRISPR-linked read assemblies. Breakpoint ranges in the reference duplicates were determined by alignment and mismatches. The example shown here is from our SV17 assembly. The two 15-kb segments have 93% similarity.
    Figure Legend Snippet: Assembly results from multiplex SV assay. ( A ) CRISPR-linked read assembly for SV1 in Assay 3 was aligned to the reference genome and compared with other long read assemblies. Red, green and blue bars indicate the portion of the assembly that aligns to the reference while a gray gap indicates no alignment. Although the gray regions have some similarity to the reference, they generally have too many homopolymer errors to successfully align. Fraction of aligned bases in the assembly is indicated at the end of the bars. ( B ) Two different sets of deletion breakpoints were determined for SV5. CRISPR-linked read SV assay captured the two SV alleles with different deletion sizes. For (A and B), the X - and Y -axes indicate the reference coordinates and the alignment of barcoded linked reads, respectively. Dashed vertical lines indicate Cas9-gRNA cut sites. ( C ) Illustration of how the breakpoints are determined in segmental duplications. Duplicated copies from GRCh38 reference genome were aligned to CRISPR-linked read assemblies. Breakpoint ranges in the reference duplicates were determined by alignment and mismatches. The example shown here is from our SV17 assembly. The two 15-kb segments have 93% similarity.

    Techniques Used: Multiplex Assay, CRISPR

    13) Product Images from "Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos"

    Article Title: Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345256

    Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange
    Figure Legend Snippet: Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange

    Techniques Used: Injection

    14) Product Images from "CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing"

    Article Title: CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

    Journal: bioRxiv

    doi: 10.1101/2020.10.13.337253

    Schematic of Cas9 Background Elimination strategy. A) Cas9 is bound to either side of target sequence. B) Off-target DNA is digested with a combination of exonucleases. C) Heat is used to dissociate that Cas9 and inactivate the exonuclieases. D) On-target fragment is available for A-tailing and sequence adapter ligation. E) Target fragments are sequenced on the MinlON for 48 hours.
    Figure Legend Snippet: Schematic of Cas9 Background Elimination strategy. A) Cas9 is bound to either side of target sequence. B) Off-target DNA is digested with a combination of exonucleases. C) Heat is used to dissociate that Cas9 and inactivate the exonuclieases. D) On-target fragment is available for A-tailing and sequence adapter ligation. E) Target fragments are sequenced on the MinlON for 48 hours.

    Techniques Used: Sequencing, Ligation

    A. gBlock assay design for Cas9 challenge with exonuclease. gBlock contained two pairs of gRNA target sites, one with PAM-out orientation and one with PAM-in orientation. Upon Cas9 binding (depicted by scissors), each set of target sites generate 3 unique fragment lengths. B. Capillary electrophoresis results from exonuclease challenge experiment with Cas9. 15nM gBlock DNA was incubated with 40nM ribonucleoprotien complex, followed by digestion with a combination of exonucleases for 2 hours. When Cas9 is used without exonucleases, the gBlock is cut to produce expected fragment lengths. Upon challenge with exonuclease, only the fragments flanked on both sides by Cas9 remain in the sample. (l=in; O=out). Wells in gel image are re-ordered for clarity.
    Figure Legend Snippet: A. gBlock assay design for Cas9 challenge with exonuclease. gBlock contained two pairs of gRNA target sites, one with PAM-out orientation and one with PAM-in orientation. Upon Cas9 binding (depicted by scissors), each set of target sites generate 3 unique fragment lengths. B. Capillary electrophoresis results from exonuclease challenge experiment with Cas9. 15nM gBlock DNA was incubated with 40nM ribonucleoprotien complex, followed by digestion with a combination of exonucleases for 2 hours. When Cas9 is used without exonucleases, the gBlock is cut to produce expected fragment lengths. Upon challenge with exonuclease, only the fragments flanked on both sides by Cas9 remain in the sample. (l=in; O=out). Wells in gel image are re-ordered for clarity.

    Techniques Used: Binding Assay, Electrophoresis, Incubation

    15) Product Images from "Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity"

    Article Title: Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27975

    MDA-MB-468 CRISPR-Cas9 generated mtp53 variant-expressing cells display thymidine sensitivity characterized by slow progression through S-phase. ( A ) The kinetics of S-phase progression of parental and CRISPR variant cell lines parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 R273H fs 387, mtp53 R273HΔ347-393, and mtp53 R273HΔ381-388 were compared post synchronization of 50% confluent cultures of each with 2 mM thymidine. At time points 0, 5, and 8 hours post release from the Thy block, cell populations from each cell line were harvested simultaneously and the cell cycle distribution of PI-stained cells was determined by flow cytometry. The distribution of cells within the G1, S, and G2/M phases at each time point is represented in the super-imposed histograms of mtp53 R273H cells (yellow), mtp53-depleted cells R273H fs 387 (gray), mtp53 R273HΔ347-393 cells (red), and mtp53 R273HΔ381-388 (green), with the percentage of G1 and G2/M cells at each time points for each cell line presented in the graphs on the right. ( B ) The abundance of the p53, MCM2 and RRM2 proteins were examined in extracts from asynchronous (–) and G1/S synchronized cell populations harvested 0, 5, and 8 hours post release from a 24 incubation with the cell cycle inhibitors aphidicolin (Aph), Thymidine (Thy), or hydroxyurea (HU). Parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 variants cell lines mtp53-depleted R273H fs 387 cells, mtp53 R273HΔ347-393 cells, and mtp53 R273HΔ381-388 cells, were cultured to 50% confluency before addition of either 5 μM Aph, 2 mM Thy, or 2 mM HU, and at the above time points cell populations were harvested and processed for either cell cycle analysis by flow cytometry, or western blotting. The distribution of cells within G1, S, and G2 based on propidium iodine (PI) staining was determined as described in
    Figure Legend Snippet: MDA-MB-468 CRISPR-Cas9 generated mtp53 variant-expressing cells display thymidine sensitivity characterized by slow progression through S-phase. ( A ) The kinetics of S-phase progression of parental and CRISPR variant cell lines parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 R273H fs 387, mtp53 R273HΔ347-393, and mtp53 R273HΔ381-388 were compared post synchronization of 50% confluent cultures of each with 2 mM thymidine. At time points 0, 5, and 8 hours post release from the Thy block, cell populations from each cell line were harvested simultaneously and the cell cycle distribution of PI-stained cells was determined by flow cytometry. The distribution of cells within the G1, S, and G2/M phases at each time point is represented in the super-imposed histograms of mtp53 R273H cells (yellow), mtp53-depleted cells R273H fs 387 (gray), mtp53 R273HΔ347-393 cells (red), and mtp53 R273HΔ381-388 (green), with the percentage of G1 and G2/M cells at each time points for each cell line presented in the graphs on the right. ( B ) The abundance of the p53, MCM2 and RRM2 proteins were examined in extracts from asynchronous (–) and G1/S synchronized cell populations harvested 0, 5, and 8 hours post release from a 24 incubation with the cell cycle inhibitors aphidicolin (Aph), Thymidine (Thy), or hydroxyurea (HU). Parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 variants cell lines mtp53-depleted R273H fs 387 cells, mtp53 R273HΔ347-393 cells, and mtp53 R273HΔ381-388 cells, were cultured to 50% confluency before addition of either 5 μM Aph, 2 mM Thy, or 2 mM HU, and at the above time points cell populations were harvested and processed for either cell cycle analysis by flow cytometry, or western blotting. The distribution of cells within G1, S, and G2 based on propidium iodine (PI) staining was determined as described in "Materials and Methods", and the percentage within G1 and G2 for each time point is represented. All extracts were analyzed by SDS-PAGE on 10% gels, and subject to western blot analysis. The experiment represented in (A) were done twice for all cell lines and in (B) they were performed twice for the CRISPR mtp53 R273H fs 387 variant and one time for all others.

    Techniques Used: Multiple Displacement Amplification, CRISPR, Generated, Variant Assay, Expressing, Blocking Assay, Staining, Flow Cytometry, Incubation, Cell Culture, Cell Cycle Assay, Western Blot, SDS Page

    Correlation of mtp53 variants protein and mRNA abundance with RRM2 and CDC7 in MDA-MB-468 CRISPR-Cas9 generated mtp53 cell lines. ( A – D ) The relative protein and mRNA abundance of TP53 , RRM2 and CDC7 was examined within asynchronous (lanes labeled (–)) and G1/S synchronized cell populations of parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 R273H fs 387, mtp53 R273HΔ347-393, and mtp53 R273HΔ381-388 by western blot analysis of total cell lysates (A), and quantitative RT-PCR analysis of mRNA B–D) prepared from each cell population. (B–D) Represent data for three independent biological replicates. A two-way anova with Dunnett’s multiple comparison was performed and the level of significance set at * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001; ns, not significant. Sub-confluent cultures (~ 50% confluent) of each cell line were synchronized at G1/S by treatment of cell populations with either 2 mM thymidine (Thy) or 2 mM hydroxyurea (HU) for 24 hours, harvested and then processed for flow cytometry (Supplementary Figure 2) and the aforementioned analyses above as described in
    Figure Legend Snippet: Correlation of mtp53 variants protein and mRNA abundance with RRM2 and CDC7 in MDA-MB-468 CRISPR-Cas9 generated mtp53 cell lines. ( A – D ) The relative protein and mRNA abundance of TP53 , RRM2 and CDC7 was examined within asynchronous (lanes labeled (–)) and G1/S synchronized cell populations of parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 R273H fs 387, mtp53 R273HΔ347-393, and mtp53 R273HΔ381-388 by western blot analysis of total cell lysates (A), and quantitative RT-PCR analysis of mRNA B–D) prepared from each cell population. (B–D) Represent data for three independent biological replicates. A two-way anova with Dunnett’s multiple comparison was performed and the level of significance set at * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001; ns, not significant. Sub-confluent cultures (~ 50% confluent) of each cell line were synchronized at G1/S by treatment of cell populations with either 2 mM thymidine (Thy) or 2 mM hydroxyurea (HU) for 24 hours, harvested and then processed for flow cytometry (Supplementary Figure 2) and the aforementioned analyses above as described in "Materials and Methods".

    Techniques Used: Multiple Displacement Amplification, CRISPR, Generated, Labeling, Western Blot, Quantitative RT-PCR, Flow Cytometry

    Defective DNA replication underscores the thymidine sensitivity of MDA-MB-468 CRISPR-Cas9 generated mtp53-depleted cells. ( A ) A direct assessment of the S-phase population was determined using flow cytometry by measuring the percentage of parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 variant mtp53-depleted C4 and C11 R273H fs 387, and mtp53 R273HΔ381-388 cells that incorporate BrdU 5 hours post release from a double Aph-Thy block. After the first synchronization at G1/S with aphidicolin, all cell populations were released into the cell cycle for 10 hours (a period sufficient for bulk genome duplication) before initiation of the second block with thymidine. Following release from either the first or second block, cells were pulse-labeled with BrdU for 30 min and then harvested either immediately thereafter in the case of samples released from a single Aph-block, or after 5 hours in the case of double-block samples labeled post-release from the Thy-block. Histograms represent the percentage of cells within each population that incorporated BrdU. ( B ) Assembly of DNA replication factors onto chromosomes of parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 mtp53-depleted R273H fs 387 cells during S-phase was measured using the chromatin fractionation assay. Cytosolic and chromatin fractions were prepared as described in
    Figure Legend Snippet: Defective DNA replication underscores the thymidine sensitivity of MDA-MB-468 CRISPR-Cas9 generated mtp53-depleted cells. ( A ) A direct assessment of the S-phase population was determined using flow cytometry by measuring the percentage of parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 variant mtp53-depleted C4 and C11 R273H fs 387, and mtp53 R273HΔ381-388 cells that incorporate BrdU 5 hours post release from a double Aph-Thy block. After the first synchronization at G1/S with aphidicolin, all cell populations were released into the cell cycle for 10 hours (a period sufficient for bulk genome duplication) before initiation of the second block with thymidine. Following release from either the first or second block, cells were pulse-labeled with BrdU for 30 min and then harvested either immediately thereafter in the case of samples released from a single Aph-block, or after 5 hours in the case of double-block samples labeled post-release from the Thy-block. Histograms represent the percentage of cells within each population that incorporated BrdU. ( B ) Assembly of DNA replication factors onto chromosomes of parental MDA-MB-468 mtp53 R273H and CRISPR-generated mtp53 mtp53-depleted R273H fs 387 cells during S-phase was measured using the chromatin fractionation assay. Cytosolic and chromatin fractions were prepared as described in "Materials and Methods" from mtp53 R273H and mtp53-depleted R273H fs 387 cell populations proliferating asynchronously (samples in lanes represent by (–)) or harvested 0, 1.25, 2.5, 5, and 7.5 hours post release from either HU- or Thy- G1/S synchronization, and then analyzed by SDS-PAGE and western blotting for the indicated proteins. The experiment in (A) was performed twice and that in (B) for two biological replicates with Thy-synchronized cells. Chromatin fractionation often resulted in multiple different migration forms of p53 protein. We think that this occurs due to do with both alternatively posttranslational modified isoforms and degradation products of p53 (but have not verified the reasons). The chromatin bound p53 showed less variation than the cytosolic p53.

    Techniques Used: Multiple Displacement Amplification, CRISPR, Generated, Flow Cytometry, Variant Assay, Blocking Assay, Labeling, Fractionation, SDS Page, Western Blot, Migration, Modification

    Domain architecture of p53 and nomenclature of clones generated via CRISPR-Cas9. ( A ) Highlights the domain architecture of p53 with emphasis on the sgRNA designed to target the oligomerization (OD) and C-terminal domain (CTD) of mtp53. ( B ) CRISPR-Cas9 was used to generate clones with either OD or CTD mutations. Clones were selected with FACS sorting of eGFP positive cells. Selected clones were named based on the region and type of mutation that resulted.
    Figure Legend Snippet: Domain architecture of p53 and nomenclature of clones generated via CRISPR-Cas9. ( A ) Highlights the domain architecture of p53 with emphasis on the sgRNA designed to target the oligomerization (OD) and C-terminal domain (CTD) of mtp53. ( B ) CRISPR-Cas9 was used to generate clones with either OD or CTD mutations. Clones were selected with FACS sorting of eGFP positive cells. Selected clones were named based on the region and type of mutation that resulted.

    Techniques Used: Clone Assay, Generated, CRISPR, FACS, Mutagenesis

    16) Product Images from "Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9"

    Article Title: Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02547-w

    Modifications of the anti-mt CRISPR treatment. Compared to the treatment shown in Fig. 1 (100X gRNA, 100X Cas9, 1 h incubation), labeled “standard”, modifications in the treatment did not show improvement. The number of peaks is comparable or even lower in the modified treatments, compared to the standard treatment. Due to the low number of reads in 6 samples, the results presented were obtained with 9.8 M reads randomly sampled. See also Supplemental Fig. S3 .
    Figure Legend Snippet: Modifications of the anti-mt CRISPR treatment. Compared to the treatment shown in Fig. 1 (100X gRNA, 100X Cas9, 1 h incubation), labeled “standard”, modifications in the treatment did not show improvement. The number of peaks is comparable or even lower in the modified treatments, compared to the standard treatment. Due to the low number of reads in 6 samples, the results presented were obtained with 9.8 M reads randomly sampled. See also Supplemental Fig. S3 .

    Techniques Used: CRISPR, Incubation, Labeling, Modification

    17) Product Images from "Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos"

    Article Title: Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345256

    Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange
    Figure Legend Snippet: Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange

    Techniques Used: Injection

    18) Product Images from "Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos"

    Article Title: Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345256

    Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange
    Figure Legend Snippet: Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange

    Techniques Used: Injection

    19) Product Images from "Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos"

    Article Title: Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345256

    Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange
    Figure Legend Snippet: Evaluation of Cas9 injection controls. ( A) DE genes across samples injected with Cas9 enzyme or Cas9 mRNA relative to uninjected batch-siblings. Plot includes the number and percentage of genes downregulated (FoldChange

    Techniques Used: Injection

    20) Product Images from "Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH)"

    Article Title: Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH)

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky411

    Schematic representation of the CATCH method. Peripheral blood mononuclear cells were embedded in an agarose gel-plug and lysed. Genomic DNA was cleaved in the plug using guided Cas9, and the target DNA was separated by PFGE. The desired band (indicated by an arrow) was excised from the gel, and the DNA was isolated, purified and analyzed.
    Figure Legend Snippet: Schematic representation of the CATCH method. Peripheral blood mononuclear cells were embedded in an agarose gel-plug and lysed. Genomic DNA was cleaved in the plug using guided Cas9, and the target DNA was separated by PFGE. The desired band (indicated by an arrow) was excised from the gel, and the DNA was isolated, purified and analyzed.

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Purification

    21) Product Images from "Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes"

    Article Title: Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz661

    CATCH targeting and linked read sequencing of HMW DNA. ( A ) Overview of the process is illustrated. First, guide RNAs target and cut multiple genomic regions of interest. Second, target HMW DNA within the specific size range is isolated by an electrophoresis-based process. At last, the target DNA is used for linked read library preparation and sequencing. The alignment of barcode linked reads shows how sequence coverage is increased across the target segment. In the alignment plot, the X -axis indicates the reference coordinates and the Y -axis shows different barcodes representing individual HMW molecules. Dashed vertical lines indicate Cas9-gRNA cut sites. ( B ) Sequencing coverage for the target regions is shown for the three assays. For Assays 1 and 2, BRCA1 -R2 and MHC-30 libraries are shown. For Assay 3, an example of a homozygous deletion (SV1) is shown. Black bars indicate the target regions. Blue and green areas in plots indicate coverage for forward and reverse reads, respectively.
    Figure Legend Snippet: CATCH targeting and linked read sequencing of HMW DNA. ( A ) Overview of the process is illustrated. First, guide RNAs target and cut multiple genomic regions of interest. Second, target HMW DNA within the specific size range is isolated by an electrophoresis-based process. At last, the target DNA is used for linked read library preparation and sequencing. The alignment of barcode linked reads shows how sequence coverage is increased across the target segment. In the alignment plot, the X -axis indicates the reference coordinates and the Y -axis shows different barcodes representing individual HMW molecules. Dashed vertical lines indicate Cas9-gRNA cut sites. ( B ) Sequencing coverage for the target regions is shown for the three assays. For Assays 1 and 2, BRCA1 -R2 and MHC-30 libraries are shown. For Assay 3, an example of a homozygous deletion (SV1) is shown. Black bars indicate the target regions. Blue and green areas in plots indicate coverage for forward and reverse reads, respectively.

    Techniques Used: Sequencing, Isolation, Electrophoresis

    Assembly results from multiplex SV assay. ( A ) CRISPR-linked read assembly for SV1 in Assay 3 was aligned to the reference genome and compared with other long read assemblies. Red, green and blue bars indicate the portion of the assembly that aligns to the reference while a gray gap indicates no alignment. Although the gray regions have some similarity to the reference, they generally have too many homopolymer errors to successfully align. Fraction of aligned bases in the assembly is indicated at the end of the bars. ( B ) Two different sets of deletion breakpoints were determined for SV5. CRISPR-linked read SV assay captured the two SV alleles with different deletion sizes. For (A and B), the X - and Y -axes indicate the reference coordinates and the alignment of barcoded linked reads, respectively. Dashed vertical lines indicate Cas9-gRNA cut sites. ( C ) Illustration of how the breakpoints are determined in segmental duplications. Duplicated copies from GRCh38 reference genome were aligned to CRISPR-linked read assemblies. Breakpoint ranges in the reference duplicates were determined by alignment and mismatches. The example shown here is from our SV17 assembly. The two 15-kb segments have 93% similarity.
    Figure Legend Snippet: Assembly results from multiplex SV assay. ( A ) CRISPR-linked read assembly for SV1 in Assay 3 was aligned to the reference genome and compared with other long read assemblies. Red, green and blue bars indicate the portion of the assembly that aligns to the reference while a gray gap indicates no alignment. Although the gray regions have some similarity to the reference, they generally have too many homopolymer errors to successfully align. Fraction of aligned bases in the assembly is indicated at the end of the bars. ( B ) Two different sets of deletion breakpoints were determined for SV5. CRISPR-linked read SV assay captured the two SV alleles with different deletion sizes. For (A and B), the X - and Y -axes indicate the reference coordinates and the alignment of barcoded linked reads, respectively. Dashed vertical lines indicate Cas9-gRNA cut sites. ( C ) Illustration of how the breakpoints are determined in segmental duplications. Duplicated copies from GRCh38 reference genome were aligned to CRISPR-linked read assemblies. Breakpoint ranges in the reference duplicates were determined by alignment and mismatches. The example shown here is from our SV17 assembly. The two 15-kb segments have 93% similarity.

    Techniques Used: Multiplex Assay, CRISPR

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    New England Biolabs cas9 protein
    Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and <t>Cas9</t> addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch
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    Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch

    Journal: Disease Markers

    Article Title: CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer

    doi: 10.1155/2018/3835783

    Figure Lengend Snippet: Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch

    Article Snippet: Next, proteinase K (2 μ g) was added, and the reaction mixture was incubated for 15 minutes at 58°C to remove the Cas9 protein.

    Techniques: Transduction, Purification, Polymerase Chain Reaction, Infection, Flow Cytometry, Cytometry, Western Blot, Expressing, Sequencing, RFLP Assay, CRISPR

    EGFR gene targeting in MDA-MB-231 cells using the CRISPR/CAS9 system: (a) schematic representation of the human EGFR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Scrambled (SC) sgRNA and EGFR sgRNA were delivered to MDA-MB-231 cells by lentivirus. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of EGFR exon 3 and exon 9. (b, c) Wild-type EGFR sequences in MDA-MB-231 cells. (d) EGFR sgRNA_1 and (e) EGFR sgRNA_2 producing a mixture of sequences around the expected Cas9 cleavage point in a pool of gene-edited cells after lentivirus transduction. TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in MDA-MB-231 cells. The pie charts show the percentages of indels in the EGFR gene edited by (f) EGFR sgRNA_1 and (g) EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and −2 indels are presented in purple and blue, respectively. The original TIDE algorithm analysis is shown for (h) EGFR sgRNA_1 and (i) EGFR sgRNA_2 virus transfected on MDA-MB-231 cells, compared to SC MDA-MB-231 cells. The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (j) The EGFR gene in MDA-MB-231 cells analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific sgRNA and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (k) Western blot analysis of EGFR protein expression. (c) Image showing parental and SC sgRNA control MDA-MB-231 cells; EGFR expression was significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced MDA-MB-231 cells.

    Journal: Disease Markers

    Article Title: CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer

    doi: 10.1155/2018/3835783

    Figure Lengend Snippet: EGFR gene targeting in MDA-MB-231 cells using the CRISPR/CAS9 system: (a) schematic representation of the human EGFR DNA locus and two protospacer sequences (blue underline) for editing. The arrowhead indicates the expected Cas9 cleavage site. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. Scrambled (SC) sgRNA and EGFR sgRNA were delivered to MDA-MB-231 cells by lentivirus. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of EGFR exon 3 and exon 9. (b, c) Wild-type EGFR sequences in MDA-MB-231 cells. (d) EGFR sgRNA_1 and (e) EGFR sgRNA_2 producing a mixture of sequences around the expected Cas9 cleavage point in a pool of gene-edited cells after lentivirus transduction. TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in MDA-MB-231 cells. The pie charts show the percentages of indels in the EGFR gene edited by (f) EGFR sgRNA_1 and (g) EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common −1 and −2 indels are presented in purple and blue, respectively. The original TIDE algorithm analysis is shown for (h) EGFR sgRNA_1 and (i) EGFR sgRNA_2 virus transfected on MDA-MB-231 cells, compared to SC MDA-MB-231 cells. The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (j) The EGFR gene in MDA-MB-231 cells analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific sgRNA and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (k) Western blot analysis of EGFR protein expression. (c) Image showing parental and SC sgRNA control MDA-MB-231 cells; EGFR expression was significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced MDA-MB-231 cells.

    Article Snippet: Next, proteinase K (2 μ g) was added, and the reaction mixture was incubated for 15 minutes at 58°C to remove the Cas9 protein.

    Techniques: Multiple Displacement Amplification, CRISPR, Activity Assay, Transduction, Infection, Purification, Sequencing, Transfection, RFLP Assay, Western Blot, Expressing