Gene editing for cancer therapy/drug discovery

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Representation of sgRNAs within the Guide-it CRISPR Genome-Wide sgRNA Library System

Representation of sgRNAs within the Guide-it CRISPR Genome-Wide sgRNA Library System. Panel A. Guide representation in plasmid library. The Brunello-based sgRNA library was cloned into the pLVXS-sgRNA-mCherry-hyg Vector and amplified. The representation of the sgRNAs within the plasmid DNA library was verified by NGS. The representation of >90% of all the stated sgRNAs within the library was within a 10-fold distribution range. Bars represent the number of sgRNAs with a specific read count. Panel B. Comparison of guide representation in the plasmid library and transduced cells. Genomic DNA was isolated from Cas9+/sgRNA+ A375 cells selected on hygromycin and PCR amplified. The PCR product was sequenced to determine read counts of each integrated sgRNA relative to the starting plasmid DNA population. We observed strong Spearman and Pearson correlations indicating that the system is able to maintain sgRNA representation in the transduced and selected cell population.

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Identification and analysis of sgRNAs isolated from cells after a 6-thioguanine screen

Identification and analysis of sgRNAs isolated from cells after a 6-thioguanine (6-TG) screen. Panel A. sgRNA representation was compared between the original plasmid library population used to make the lentivirus preparation and the resulting transduced, selected and non-selected cell populations. All four sgRNAs for HPRT (red dots) and NUDT5 (black dots) were enriched in the 6-TG-selected population (blue dots). The representation of these sgRNAs in the plasmid library (gray) and transduced, non-selected population (orange) is also shown. Panel B. After selection, the sgRNA representation was shifted in response (blue) to 6-TG. Individual sgRNAs that were enriched are highlighted within this correlation (red and orange dots). Inset shows fold-enrichment of the four sgRNAs for the HPRT gene as compared to the non-selected population.

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Workflow schematic for an sgRNA library screen using 6-thioguanine (6-TG) selection

Workflow schematic for an sgRNA library screen using 6-thioguanine (6-TG) selection. After transduction of Cas9+ A375 cells with the sgRNA library, hygromycin selection, and expansion, the Cas9+/ sgRNA+ cells were split into two populations. A part of the Cas9+/sgRNA+ cell population was exposed to 6-TG, while the control population was expanded under normal culture conditions, without adding 6-TG. After expansion of both cell populations, genomic DNA was harvested and prepared for sequencing with the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit.

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Determination of transduction efficiency of the sgRNA library lentivirus using mCherry fluorescence

Determination of transduction efficiency of the sgRNA library lentivirus using mCherry fluorescence. Lentivirus containing the sgRNA library was produced following the instructions in the user manual. Lentivirus was harvested after 48 hrs and used to transduce Cas9-expressing A375 cells at varying MOIs. Transduced cells were plated and analyzed for transduction efficiency by fluorescence microscopy and FACS after 48 hours.

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Vector and sgRNA scaffold design used in the Guide-it CRISPR sgRNA library

Vector and sgRNA scaffold design used in the Guide-it CRISPR sgRNA library. Panel A. The pLVXS-EF1a-Cas9-PGK-Puro and pLVXS-sgRNA-mCherry-hyg vectors maps showing the lentiviral vector backbone. The vectors are self-inactivating for increased safety during production and use. The pLVXS-sgRNA-mCherry-hyg vector contains both mCherry and hygromycin markers expressed from an IRES-linked bicistronic expression cassette. The sgRNAs are expressed from a human U6 promoter and contain an optimized scaffold sequence for better Cas9 loading and editing efficiency (Panel B).

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Identification of insertions and deletions (indels) in the CD81 gene after CRISPR/Cas9 targeting

Identification of insertions and deletions (indels) in the CD81 gene after CRISPR/Cas9 targeting. HeLa cells were transfected with plasmids encoding Cas9 and an sgRNA targeting the CD81 gene. The Guide-it Indel Identification Kit was used to prepare CD81 target sites for DNA sequence analysis. The sequencing data from six clones was aligned with the wild-type sequence, revealing a broad range of indels in the CD81 gene.

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The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety of insertions and deletions (indels) introduced by nuclease-based genome editing

The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety of insertions and deletions (indels) introduced by nuclease-based genome editing. The protocol uses direct PCR to amplify a genomic DNA fragment (~500 to 700 bp) containing the target site directly from crude cell lysates (step 1). The resulting amplified fragments contain a pool of edited target sites from individual cells. These PCR products are cloned directly into a pre-linearized pUC19 vector using the In-Fusion Cloning system (step 2). After transformation of an optimized E. coli strain, colony PCR is used to amplify the target site from the plasmid (step 3). DNA sequencing is then used to identify the different indels generated at the targeted genomic site (step 4)

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The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).

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FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci

FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci. Cas9-sgRNA RNPs targeting SEC61B and TUBA1A and corresponding AcGFP1 ssDNA homology-directed repair (HDR) templates generated using the Guide-it Long ssDNA Production System were electroporated into ChiPSC18 cells. Following culturing, electroporated target cells and non-electroporated negative control cells were analyzed for GFP expression by FACS. Using this approach, knockin efficiencies of 12.55% and 2.75% were observed for insertion of AcGFP1 at SEC61B and TUBA1A, respectively.

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PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS

PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS. ChiPSC18 cell populations were electroporated with Cas9-sgRNA RNPs and sense or antisense ssDNA HDR templates (labeled ssDNA-1 or ssDNA-2, respectively) that were designed for knockin of AcGFP1 at the SEC61B locus. The cells were sorted on the basis of GFP expression via FACS. Individual clones from the GFP-positive populations were isolated by limiting dilution and cultured to yield clonal cell populations. Genomic DNA extracted from the clonal populations was analyzed by PCR using primers (L-F and R-R, respectively) designed to anneal on either side of the GFP insertion site as shown in the schematic. The GFP and homology arm sequences included in the ssDNA templates are shown in green and light blue, respectively, while the genomic sequences outside the region targeted by the ssDNA homology arms are shown in dark blue. The PCR primers were designed so that the presence of the inserted AcGFP1 sequence yielded a 1.885-kb PCR product, while absence of the AcGFP1 insert yielded a 1.159-kb PCR product. Gel electrophoresis of PCR products confirmed the occurrence of monoallelic knockins of AcGFP1 at SEC61B, represented by double bands (ssDNA-1 Lane C5, and ssDNA-2 Lane C15), and biallelic knockins of AcGFP1 at SEC61B, represented by single bands (ssDNA-1 Lanes C1, C7, C8, C11, and ssDNA-2 Lanes C2, C6, C8, and C10). Two clones lacking any copies of the AcGFP1 insert were also identified (ssDNA-1 Lane C10, and ssDNA-2 Lane C11).

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Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus

Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus. The ssDNA construct generated using the Guide-it Long ssDNA Production System included the AcGFP1 coding sequence (~700 nt in length) flanked on either side by homology arms ~350 nt in length. The homology arms were designed to insert the AcGFP1 sequence immediately 5' relative to Exon 1 of SEC61B. As indicated, the ssDNA construct did not include a promoter sequence, so that expression of the resulting AcGFP1-SEC61B fusion protein would be driven by the endogenous SEC61B promoter. Binding sites for PCR primers used to detect the inserted AcGFP1 sequence are shown by blue arrows.

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Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus

Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus. The ssDNA construct generated using the Guide-it Long ssDNA Production System included the AcGFP1 coding sequence (~700 nt in length) flanked on either side by homology arms ~350 nt in length. The homology arms were designed to insert the AcGFP1 sequence at the 5' end of Exon 2 of TUBA1A. As indicated, the ssDNA construct did not include a promoter sequence, so that expression of the resulting AcGFP1-TUBA1A fusion protein would be driven by the endogenous TUBA1A promoter.

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Toxicity comparison for electroporation of dsDNA or ssDNA

Toxicity comparison for electroporation of dsDNA or ssDNA. ChiPSC18 cells were electroporated with Cas9-sgRNA RNPs targeting the TUBA1A locus, in the absence or presence of homology-directed repair (HDR) templates consisting of dsDNA or ssDNA. After 48 hours of culturing, imaging was performed to compare the toxicity associated with each HDR template. While electroporation of either DNA template resulted in increased toxicity compared to electroporation of Cas9-sgRNA RNPs alone (as evidenced by lower cell counts following culturing), application of the ssDNA template resulted in greatly reduced toxicity compared to the dsDNA template.

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In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates. ChiPSC18 cells were edited using Cas9-sgRNAs and ssDNA HDR templates designed for knockin of AcGFP1 at the SEC61B and TUBA1A loci, respectively. GFP-positive clonal cell lines obtained from each edited population were then stained with DAPI and analyzed by fluorescence microscopy. Expression of GFP-SEC61B was observed in endosomal compartments (left), while GFP-TUBA1A localized to microtubules (right).

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The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA

The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA. In the first step your target sequence is amplified directly from your target cells using the Terra PCR Direct Polymerase included in the kit, so there is no need to extract and purify genomic DNA from your cell population prior to amplification of your target sequence. The amplicon is then melted and hybridized to form the mismatched targets that can be cleaved by the Guide-it Resolvase.

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Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells

Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells. 293T cells were transfected with plasmids encoding Cas9 and a sgRNA specific for the AAVS1 locus. Transfected cells were harvested 48 hr post-transfection and mixed with untransfected cells at varying ratios. An amplicon containing the targeted AAVS1 locus was generated using Terra Direct Polymerase Mix, and the PCR products were purified and cleaved using either Guide-it Resolvase or the Cel1 enzyme (Surveyor assay). Mutations were easily discernable when using the Guide-it kit. In contrast, the Surveyor Assay showed considerable smearing, making it difficult to determine cleavage efficiency and reducing the ability to detect low levels of mutation.

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Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system

Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system. Panel A. Schematic of the AcGFP DNA sequence and the location of sgRNAs tested and primer placement for the mutation detection assay. HT1080 cells containing a single copy of AcGFP1 were transfected with 1.5 μg of plasmid DNA for Cas9 expression and 1.5 μg of a plasmid harboring one of two sgRNAs (T1 or T2) using Xfect Transfection Reagent. The cell population was assayed 6 days post-transfection for cleavage efficiency and loss of fluorescence. Panel B. Using the Guide-it Mutation Detection Kit, cleavage products were detected for both sgRNAs, indicating that both CRISPRs successfully disrupted the AcGFP1 locus. Panel C. The AcGFP1 disruptions were functionally relevant, as a subpopulation of non-fluorescent cells could be detected by FACS.

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631448: Guide-it Mutation Detection Kit

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Knock out of CD81 in induced pluripotent stem cells

Knock out of CD81 in induced pluripotent stem cells. In this experiment, two induced pluripotent stem cell lines, hiPSC-18 and hiPSC-22, were electroporated withCas9/sgRNA RNPs against CD81. Cells were stained for CD81 and then run on a FACS machine to detect CD81 ten days after editing. The negative staining control shows the FACS data from cells without antibody staining, while the electroporation control shows cells electroporated without Cas9/sgRNA RNPs. When cells were electroporated with both Cas9 and sgRNA, very high editing efficiency was observed.

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Homology-directed repair at the AAVS1 or CXCR4 genes

Homology-directed repair at the AAVS1 or CXCR4 genes. Panel A. These diagrams demonstrate how the HDR experiments in CD34+ HSCs were done. A ssdNA template containing a HindIII restriction site was inserted into the AAVS1 gene, and a template containing both HindIII and BamHI restriction sites was inserted into the CXCR4 gene. The homology arms were 90 bp on each side. Panel B. After electroporation of the repair template and Cas9/sgRNA RNPs, gene targets were amplified from target cells using PCR and analyzed by restriction digest. Editing efficiencies are shown above each positive well on the gel. The extra band in the BamHI digest of the CXCR4 gene is due to a second BamHI site already present in the wild-type gene before editing. We verified that 98% of the HSCs stained positive for CD34+ five days after editing.

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Gene knockouts in the Cellartis hiPSC-18 cell line

Gene knockouts in the Cellartis hiPSC-18 cell line. The strength of the cleaved bands gives a semiquantitative estimate of the percentage of edited cells. As can be seen, the Guide-it recombinant Cas9, when combined with sgRNA from the Guide-it In Vitro Transcription Kit, provides consistent and effective gene editing for many targets when compared with other vendors’ recommended methods for producing guide RNAs. Numbers at the bottom of each gel represent gene editing efficiencies (expressed as a %) for the indicated RNPs.

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632641: Guide-it Recombinant Cas9 (Electroporation-Ready)