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    Structured Review

    Thermo Fisher bsmbi
    Design, delivery, and selection of ScanDel library of programmed deletions for identification of non-coding regulatory elements. A) gRNA pairs are designed from a filtered set of protospacers from all Cas9 PAM sequences (5’-NGGs) in the HPRT1 locus (see also Fig. 2A ). Sites that are > 25 bp apart and > 50 bp away from exons with on-target efficiency and off-target scores above thresholds are kept. Any spacers with <t>BsmBI</t> restriction enzyme sites or predicted to have off-target hits in other 6TG resistance genes or in KBM7 essential genes (the HAP1 parental cell line) are excluded. Tiles are designed by pairing each remaining spacer to two downstream spacers targeting sequence ∼1 Kb away and ∼2 Kb away. This results in high redundancy of independently programmed, overlapping deletions across the locus (see also Fig. 2B ). B ) All spacer pairs that correspond to programmed deletions are synthesized on a microarray ( inset ). Each spacer is also synthesized as a self-pair as a control for its independent effects. If a self-paired spacer scores positively in the screen, any pairs that use that spacer are removed from analysis ( Fig. S2 ). U6 and gRNA backbone sequence flank the spacer pairs for Gibson-mediated cloning into <t>lentiGuide-Puro</t> ( Sanjana, Shalem, Zhang, 2014 ), and mirrored BsmBI cut sites separate the spacer pairs to facilitate insertion of a second gRNA backbone and the H1 promoter ( beige ). In the final library, each gRNA is expressed from its own PolIII promoter. This design facilitates PCR and direct sequencing-based quantification of gRNA pair abundances. C) The lentiviral library of gRNA pairs is cloned at a minimum of 20x coverage (relative to library complexity) and transduced into HAP1 cells stably expressing Cas9 (via lentiCas9-Blast ( Sanjana et al., 2014 )) at low MOI. After a week of puromycin selection, the cells are sampled to measure the baseline abundance of each gRNA pair. The final cell population is harvested after a week of 6-thioguanine (6TG) treatment, which selects for cells that have lost HPRT enzymatic function. The phenotypic prevalence of each programmed deletion is quantified by PCR and deep sequencing of the gRNA pairs before and after selection.
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    1) Product Images from "Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements"

    Article Title: Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

    Journal: bioRxiv

    doi: 10.1101/092445

    Design, delivery, and selection of ScanDel library of programmed deletions for identification of non-coding regulatory elements. A) gRNA pairs are designed from a filtered set of protospacers from all Cas9 PAM sequences (5’-NGGs) in the HPRT1 locus (see also Fig. 2A ). Sites that are > 25 bp apart and > 50 bp away from exons with on-target efficiency and off-target scores above thresholds are kept. Any spacers with BsmBI restriction enzyme sites or predicted to have off-target hits in other 6TG resistance genes or in KBM7 essential genes (the HAP1 parental cell line) are excluded. Tiles are designed by pairing each remaining spacer to two downstream spacers targeting sequence ∼1 Kb away and ∼2 Kb away. This results in high redundancy of independently programmed, overlapping deletions across the locus (see also Fig. 2B ). B ) All spacer pairs that correspond to programmed deletions are synthesized on a microarray ( inset ). Each spacer is also synthesized as a self-pair as a control for its independent effects. If a self-paired spacer scores positively in the screen, any pairs that use that spacer are removed from analysis ( Fig. S2 ). U6 and gRNA backbone sequence flank the spacer pairs for Gibson-mediated cloning into lentiGuide-Puro ( Sanjana, Shalem, Zhang, 2014 ), and mirrored BsmBI cut sites separate the spacer pairs to facilitate insertion of a second gRNA backbone and the H1 promoter ( beige ). In the final library, each gRNA is expressed from its own PolIII promoter. This design facilitates PCR and direct sequencing-based quantification of gRNA pair abundances. C) The lentiviral library of gRNA pairs is cloned at a minimum of 20x coverage (relative to library complexity) and transduced into HAP1 cells stably expressing Cas9 (via lentiCas9-Blast ( Sanjana et al., 2014 )) at low MOI. After a week of puromycin selection, the cells are sampled to measure the baseline abundance of each gRNA pair. The final cell population is harvested after a week of 6-thioguanine (6TG) treatment, which selects for cells that have lost HPRT enzymatic function. The phenotypic prevalence of each programmed deletion is quantified by PCR and deep sequencing of the gRNA pairs before and after selection.
    Figure Legend Snippet: Design, delivery, and selection of ScanDel library of programmed deletions for identification of non-coding regulatory elements. A) gRNA pairs are designed from a filtered set of protospacers from all Cas9 PAM sequences (5’-NGGs) in the HPRT1 locus (see also Fig. 2A ). Sites that are > 25 bp apart and > 50 bp away from exons with on-target efficiency and off-target scores above thresholds are kept. Any spacers with BsmBI restriction enzyme sites or predicted to have off-target hits in other 6TG resistance genes or in KBM7 essential genes (the HAP1 parental cell line) are excluded. Tiles are designed by pairing each remaining spacer to two downstream spacers targeting sequence ∼1 Kb away and ∼2 Kb away. This results in high redundancy of independently programmed, overlapping deletions across the locus (see also Fig. 2B ). B ) All spacer pairs that correspond to programmed deletions are synthesized on a microarray ( inset ). Each spacer is also synthesized as a self-pair as a control for its independent effects. If a self-paired spacer scores positively in the screen, any pairs that use that spacer are removed from analysis ( Fig. S2 ). U6 and gRNA backbone sequence flank the spacer pairs for Gibson-mediated cloning into lentiGuide-Puro ( Sanjana, Shalem, Zhang, 2014 ), and mirrored BsmBI cut sites separate the spacer pairs to facilitate insertion of a second gRNA backbone and the H1 promoter ( beige ). In the final library, each gRNA is expressed from its own PolIII promoter. This design facilitates PCR and direct sequencing-based quantification of gRNA pair abundances. C) The lentiviral library of gRNA pairs is cloned at a minimum of 20x coverage (relative to library complexity) and transduced into HAP1 cells stably expressing Cas9 (via lentiCas9-Blast ( Sanjana et al., 2014 )) at low MOI. After a week of puromycin selection, the cells are sampled to measure the baseline abundance of each gRNA pair. The final cell population is harvested after a week of 6-thioguanine (6TG) treatment, which selects for cells that have lost HPRT enzymatic function. The phenotypic prevalence of each programmed deletion is quantified by PCR and deep sequencing of the gRNA pairs before and after selection.

    Techniques Used: Selection, Sequencing, Synthesized, Microarray, Clone Assay, Polymerase Chain Reaction, Stable Transfection, Expressing

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    Thermo Fisher esp3i bsmbi 10 u µl
    Principle of primer design and cloning for LentiCRISPR-Cas9 mediated gene knockout. for sgRNA binding sites. B. After choosing a guide by score and location, complement primer sequence can be generated. C and D. For ligation into the Esp <t>3I</t> restriction site of the pLentiCRISPR plasmid sticky ends (CACC/CAAA) and the U6 transcriptional start site (TSS), (G) has to be added to the oligonucleotide sequence. U6, RNA Pol III promoter; EFS, EF1 short promoter.
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    96
    Thermo Fisher esp3i
    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after <t>Esp3I</t> treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
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    94
    Thermo Fisher bsmbi
    Design, delivery, and selection of ScanDel library of programmed deletions for identification of non-coding regulatory elements. A) gRNA pairs are designed from a filtered set of protospacers from all Cas9 PAM sequences (5’-NGGs) in the HPRT1 locus (see also Fig. 2A ). Sites that are > 25 bp apart and > 50 bp away from exons with on-target efficiency and off-target scores above thresholds are kept. Any spacers with <t>BsmBI</t> restriction enzyme sites or predicted to have off-target hits in other 6TG resistance genes or in KBM7 essential genes (the HAP1 parental cell line) are excluded. Tiles are designed by pairing each remaining spacer to two downstream spacers targeting sequence ∼1 Kb away and ∼2 Kb away. This results in high redundancy of independently programmed, overlapping deletions across the locus (see also Fig. 2B ). B ) All spacer pairs that correspond to programmed deletions are synthesized on a microarray ( inset ). Each spacer is also synthesized as a self-pair as a control for its independent effects. If a self-paired spacer scores positively in the screen, any pairs that use that spacer are removed from analysis ( Fig. S2 ). U6 and gRNA backbone sequence flank the spacer pairs for Gibson-mediated cloning into <t>lentiGuide-Puro</t> ( Sanjana, Shalem, Zhang, 2014 ), and mirrored BsmBI cut sites separate the spacer pairs to facilitate insertion of a second gRNA backbone and the H1 promoter ( beige ). In the final library, each gRNA is expressed from its own PolIII promoter. This design facilitates PCR and direct sequencing-based quantification of gRNA pair abundances. C) The lentiviral library of gRNA pairs is cloned at a minimum of 20x coverage (relative to library complexity) and transduced into HAP1 cells stably expressing Cas9 (via lentiCas9-Blast ( Sanjana et al., 2014 )) at low MOI. After a week of puromycin selection, the cells are sampled to measure the baseline abundance of each gRNA pair. The final cell population is harvested after a week of 6-thioguanine (6TG) treatment, which selects for cells that have lost HPRT enzymatic function. The phenotypic prevalence of each programmed deletion is quantified by PCR and deep sequencing of the gRNA pairs before and after selection.
    Bsmbi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Principle of primer design and cloning for LentiCRISPR-Cas9 mediated gene knockout. for sgRNA binding sites. B. After choosing a guide by score and location, complement primer sequence can be generated. C and D. For ligation into the Esp 3I restriction site of the pLentiCRISPR plasmid sticky ends (CACC/CAAA) and the U6 transcriptional start site (TSS), (G) has to be added to the oligonucleotide sequence. U6, RNA Pol III promoter; EFS, EF1 short promoter.

    Journal: Bio-protocol

    Article Title: Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

    doi: 10.21769/BioProtoc.2222

    Figure Lengend Snippet: Principle of primer design and cloning for LentiCRISPR-Cas9 mediated gene knockout. for sgRNA binding sites. B. After choosing a guide by score and location, complement primer sequence can be generated. C and D. For ligation into the Esp 3I restriction site of the pLentiCRISPR plasmid sticky ends (CACC/CAAA) and the U6 transcriptional start site (TSS), (G) has to be added to the oligonucleotide sequence. U6, RNA Pol III promoter; EFS, EF1 short promoter.

    Article Snippet: Esp 3I restriction enzyme (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER0451).

    Techniques: Clone Assay, Gene Knockout, Binding Assay, Sequencing, Generated, Ligation, Plasmid Preparation

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Journal: Nucleic Acids Research

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    doi: 10.1093/nar/gkaa270

    Figure Lengend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Article Snippet: To effect Esp3I-digestion (e.g. for step iii in Figure ), beads were then incubated for 2 h at 37°C, while shaking at 1200 RPM, in a solution of 200 units of Esp3I, 1 mM DTT, in 1× Tango buffer (ThermoFisher Scientific, in a total volume of 120 μl.

    Techniques: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    Design and workflow of a SpliMLiB library for Z IgE . ( A ) Model structure for Z IgE (modelled by Swissmodel ( 118 ), based on a template with PDB ID 2m5a ( 119 ), indicating the locations of the four positions targeted in the SpliMLiB library. ( B ) Schematic overview of the final Z IgE expression construct that was assembled in four SpliMLiB attachment-rounds. The Z IgE sequence was divided into four sets of fragments, each of which carried one of the targeted positions. These SpliMLiB input fragments were generated either by PCR (fragment sets frag T10 frag M35 ) or through annealing of partially complementary oligonucleotides (fragment sets frag M18 frag G28 ). The first set of fragments to be immobilised, frag M35 , was functionalised with DBCO, allowing immobilisation of fragments through copper-free click chemistry to azide-functionalised beads. The last set of fragments to be ligated, frag T10 , was functionalised with FAM, allowing monitoring of the efficiency of total SpliMLiB library assembly efficiency. The Esp3I type IIs sites included on the ends of the PCR-generated fragments supported seamless ligations to the oligonucleotide duplexes which had 5′-overhangs by design and which had been enzymatically 5′-phosphorylated. ( C ) The SpliMLiB workflow is schematically depicted. In a first attachment-round, DNA was immobilised on split populations of beads using copper-free click chemistry (i), before beads were mixed (ii) and subjected to an on-bead restriction reaction (iii) in order to generate a 5′-overhang. Next, beads were split again and 5′-phosphorylated synthetic duplex DNA with a 5′-overhang complementary to the 5′-overhang (generated in step iii) was ligated to the bead-immobilised DNA. After subsequent mixing (v) and splitting of the beads, the bead-bound DNA was ready for extension by yet another 5′-phosphorylated synthetic duplex DNA fragment (vi). Beads were then mixed (vii) and split for the final ligation (viii) to add a PCR fragment carrying a 5′-overhang (generated by off-bead type IIs restriction), complementary to the penultimate fragment, the 5′-phosphorylated synthetic duplex DNA. Each PCR amplicon from this last set of fragments was labelled with a 5′-FAM at the far end, for flow cytometric analysis of the mixed final library (ix). ( D ) The efficiency of SpliMLiB library construction was analysed by flow cytometry. The positive control (PC) was prepared by immobilising the full length Z IgE DNA fragment by click chemistry on the beads (identically end-labelled with fluorescein as the library bead DNA). Untreated beads that did not contain any DNA served as the negative control (NC).

    Journal: Nucleic Acids Research

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    doi: 10.1093/nar/gkaa270

    Figure Lengend Snippet: Design and workflow of a SpliMLiB library for Z IgE . ( A ) Model structure for Z IgE (modelled by Swissmodel ( 118 ), based on a template with PDB ID 2m5a ( 119 ), indicating the locations of the four positions targeted in the SpliMLiB library. ( B ) Schematic overview of the final Z IgE expression construct that was assembled in four SpliMLiB attachment-rounds. The Z IgE sequence was divided into four sets of fragments, each of which carried one of the targeted positions. These SpliMLiB input fragments were generated either by PCR (fragment sets frag T10 frag M35 ) or through annealing of partially complementary oligonucleotides (fragment sets frag M18 frag G28 ). The first set of fragments to be immobilised, frag M35 , was functionalised with DBCO, allowing immobilisation of fragments through copper-free click chemistry to azide-functionalised beads. The last set of fragments to be ligated, frag T10 , was functionalised with FAM, allowing monitoring of the efficiency of total SpliMLiB library assembly efficiency. The Esp3I type IIs sites included on the ends of the PCR-generated fragments supported seamless ligations to the oligonucleotide duplexes which had 5′-overhangs by design and which had been enzymatically 5′-phosphorylated. ( C ) The SpliMLiB workflow is schematically depicted. In a first attachment-round, DNA was immobilised on split populations of beads using copper-free click chemistry (i), before beads were mixed (ii) and subjected to an on-bead restriction reaction (iii) in order to generate a 5′-overhang. Next, beads were split again and 5′-phosphorylated synthetic duplex DNA with a 5′-overhang complementary to the 5′-overhang (generated in step iii) was ligated to the bead-immobilised DNA. After subsequent mixing (v) and splitting of the beads, the bead-bound DNA was ready for extension by yet another 5′-phosphorylated synthetic duplex DNA fragment (vi). Beads were then mixed (vii) and split for the final ligation (viii) to add a PCR fragment carrying a 5′-overhang (generated by off-bead type IIs restriction), complementary to the penultimate fragment, the 5′-phosphorylated synthetic duplex DNA. Each PCR amplicon from this last set of fragments was labelled with a 5′-FAM at the far end, for flow cytometric analysis of the mixed final library (ix). ( D ) The efficiency of SpliMLiB library construction was analysed by flow cytometry. The positive control (PC) was prepared by immobilising the full length Z IgE DNA fragment by click chemistry on the beads (identically end-labelled with fluorescein as the library bead DNA). Untreated beads that did not contain any DNA served as the negative control (NC).

    Article Snippet: To effect Esp3I-digestion (e.g. for step iii in Figure ), beads were then incubated for 2 h at 37°C, while shaking at 1200 RPM, in a solution of 200 units of Esp3I, 1 mM DTT, in 1× Tango buffer (ThermoFisher Scientific, in a total volume of 120 μl.

    Techniques: Expressing, Construct, Sequencing, Generated, Polymerase Chain Reaction, Ligation, Amplification, Flow Cytometry, Positive Control, Negative Control

    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).

    Journal: Nucleic Acids Research

    Article Title: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

    doi: 10.1093/nar/gkr218

    Figure Lengend Snippet: Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).

    Article Snippet: Finally, into this plasmid, the XbaI–SacI fragment of pTAL3 containing the TALEN backbone construct was introduced at the corresponding sites. pTAL1 was created by replacing the SphI fragment of tal1C in pCS691 with the corresponding SphI fragment of pTAL3, containing the lacZ gene and the Esp3I sites and flanking sequences for accepting final arrays. pCS691 is a derivative of Gateway entry vector pENTR-D (Invitrogen) containing between the attL sites, the complete tal1c gene preceded by both Kozak and Shine–Dalgarno consensus sequences for efficient translation in eukaryotic or bacterial cells, respectively.

    Techniques: Construct, Plasmid Preparation, Activation Assay, Clone Assay, Subcloning, Sequencing

    Design, delivery, and selection of ScanDel library of programmed deletions for identification of non-coding regulatory elements. A) gRNA pairs are designed from a filtered set of protospacers from all Cas9 PAM sequences (5’-NGGs) in the HPRT1 locus (see also Fig. 2A ). Sites that are > 25 bp apart and > 50 bp away from exons with on-target efficiency and off-target scores above thresholds are kept. Any spacers with BsmBI restriction enzyme sites or predicted to have off-target hits in other 6TG resistance genes or in KBM7 essential genes (the HAP1 parental cell line) are excluded. Tiles are designed by pairing each remaining spacer to two downstream spacers targeting sequence ∼1 Kb away and ∼2 Kb away. This results in high redundancy of independently programmed, overlapping deletions across the locus (see also Fig. 2B ). B ) All spacer pairs that correspond to programmed deletions are synthesized on a microarray ( inset ). Each spacer is also synthesized as a self-pair as a control for its independent effects. If a self-paired spacer scores positively in the screen, any pairs that use that spacer are removed from analysis ( Fig. S2 ). U6 and gRNA backbone sequence flank the spacer pairs for Gibson-mediated cloning into lentiGuide-Puro ( Sanjana, Shalem, Zhang, 2014 ), and mirrored BsmBI cut sites separate the spacer pairs to facilitate insertion of a second gRNA backbone and the H1 promoter ( beige ). In the final library, each gRNA is expressed from its own PolIII promoter. This design facilitates PCR and direct sequencing-based quantification of gRNA pair abundances. C) The lentiviral library of gRNA pairs is cloned at a minimum of 20x coverage (relative to library complexity) and transduced into HAP1 cells stably expressing Cas9 (via lentiCas9-Blast ( Sanjana et al., 2014 )) at low MOI. After a week of puromycin selection, the cells are sampled to measure the baseline abundance of each gRNA pair. The final cell population is harvested after a week of 6-thioguanine (6TG) treatment, which selects for cells that have lost HPRT enzymatic function. The phenotypic prevalence of each programmed deletion is quantified by PCR and deep sequencing of the gRNA pairs before and after selection.

    Journal: bioRxiv

    Article Title: Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

    doi: 10.1101/092445

    Figure Lengend Snippet: Design, delivery, and selection of ScanDel library of programmed deletions for identification of non-coding regulatory elements. A) gRNA pairs are designed from a filtered set of protospacers from all Cas9 PAM sequences (5’-NGGs) in the HPRT1 locus (see also Fig. 2A ). Sites that are > 25 bp apart and > 50 bp away from exons with on-target efficiency and off-target scores above thresholds are kept. Any spacers with BsmBI restriction enzyme sites or predicted to have off-target hits in other 6TG resistance genes or in KBM7 essential genes (the HAP1 parental cell line) are excluded. Tiles are designed by pairing each remaining spacer to two downstream spacers targeting sequence ∼1 Kb away and ∼2 Kb away. This results in high redundancy of independently programmed, overlapping deletions across the locus (see also Fig. 2B ). B ) All spacer pairs that correspond to programmed deletions are synthesized on a microarray ( inset ). Each spacer is also synthesized as a self-pair as a control for its independent effects. If a self-paired spacer scores positively in the screen, any pairs that use that spacer are removed from analysis ( Fig. S2 ). U6 and gRNA backbone sequence flank the spacer pairs for Gibson-mediated cloning into lentiGuide-Puro ( Sanjana, Shalem, Zhang, 2014 ), and mirrored BsmBI cut sites separate the spacer pairs to facilitate insertion of a second gRNA backbone and the H1 promoter ( beige ). In the final library, each gRNA is expressed from its own PolIII promoter. This design facilitates PCR and direct sequencing-based quantification of gRNA pair abundances. C) The lentiviral library of gRNA pairs is cloned at a minimum of 20x coverage (relative to library complexity) and transduced into HAP1 cells stably expressing Cas9 (via lentiCas9-Blast ( Sanjana et al., 2014 )) at low MOI. After a week of puromycin selection, the cells are sampled to measure the baseline abundance of each gRNA pair. The final cell population is harvested after a week of 6-thioguanine (6TG) treatment, which selects for cells that have lost HPRT enzymatic function. The phenotypic prevalence of each programmed deletion is quantified by PCR and deep sequencing of the gRNA pairs before and after selection.

    Article Snippet: First, the lentiGuide-Puro backbone (Addgene #52963) is digested with BsmBI (FastDigest Esp3I, Thermo) and gel purified.

    Techniques: Selection, Sequencing, Synthesized, Microarray, Clone Assay, Polymerase Chain Reaction, Stable Transfection, Expressing