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Tech Note

Performing a phenotypic screen using the Guide-it CRISPR Genome-Wide sgRNA Library System

Guide-it CRISPR Genome-Wide sgRNA Library System

  • Pre-validated sgRNA representation is maintained among transduced cells
  • Design principles of the Brunello library are combined with an optimized expression vector and sgRNA-scaffold design to maximize editing efficiency and minimize off-target effects
  • Easy-to-use, lyophilized formulation of Cas9 and sgRNA packaging mixes combined with mCherry reporter simplify preparation and titration of viral particles
  • 100% editing efficiency confirmed in all randomly selected clones from a Cas9+/sgRNA+ cell population
  • Implementation of this system in a functional screen correctly identified genes involved in the purine salvage pathway
Introduction Library design features Results Conclusions Methods

Introduction  

Whole-genome screens employing CRISPR/Cas9 technology have been recently demonstrated, granting researchers the ability to quickly identify novel gene targets involved in biological pathways of interest (Doench, 2018). CRISPR/Cas9-based screens improve on previous methods, such as RNAi, by providing the ability to completely knock out—rather than knock down—gene function in successfully edited cells, yielding more robust phenotypes that are easier to detect. With the aim of providing a highly effective, user-friendly system for performing genome-wide phenotypic screens in human cell lines, we have applied the latest research regarding sgRNA library design and our expertise in lentiviral systems to develop the Guide-it CRISPR Genome-Wide sgRNA Library System.

Library design features  

Phenotypic screens using Guide-it CRISPR/Cas9 involve many important processes that determine their effectiveness in identifying novel genes. These include the size of the sgRNA library, the design and selection of individual single guide RNAs (sgRNAs), and optimization of plasmid library amplification, lentiviral packaging, and target-cell transduction processes.

Our library consists of >76,000 sgRNAs targeting ~19,000 protein-coding genes, with four highly active sgRNAs per target (Doench et al. 2016). While it is possible to employ larger libraries, it can be difficult to maintain representation of each sgRNA during amplification as the number of sgRNAs increases. For this reason, some libraries are split into modules, requiring that multiple plasmid amplification, lentiviral packaging, and target-cell transduction steps be performed in parallel, increasing the possibility of bias or loss of sgRNA representation.

The sgRNAs included in our library were chosen from the Brunello library (Doench et al. 2016), which have been shown to have better target specificity with minimal off-target effects to provide an increased probability of identifying relevant genes as compared to other available libraries.

sgRNA representation is one important metric that determines library performance during a screen. An ideal library should have every single sgRNA represented at the exact same quantity, but this is impossible in practice due to variations in sgRNA synthesis and plasmid library amplification. The Guide-it CRISPR Genome-Wide sgRNA Library was synthesized and amplified using our decades of experience working with cDNA libraries to optimize the process, resulting in a very tight distribution of sgRNAs. Each lot is analyzed to confirm that >90% of all sgRNAs in the library are within a 10-fold distribution range (Figure 1, Panel A; sign up to download complete sgRNA representation data).

By providing the library in an easy-to-use format that simplifies lentiviral production and target-cell transduction, our system helps the user maintain sgRNA representation in the transduced cell population without having to deal with the tedious library amplification and virus optimization steps (Figure 1, Panel B).

sgRNA representation

Figure 1. Representation of sgRNAs within the Guide-it CRISPR Genome-Wide sgRNA Library System. Panel A. sgRNA representation in the starting plasmid population. The Brunello-based sgRNA library was cloned into the pLVXS-sgRNA-mCherry-hyg Vector and amplified. Representation of the sgRNAs within the plasmid DNA was verified by next-generation sequencing (NGS). Bars represent the number of sgRNAs detected at a given read count within the population. Panel B. The correlation between read counts of each integrated sgRNA for the transduced cell population relative to read counts of the corresponding sgRNA for the starting plasmid population. These data show strong Spearman and Pearson correlations, indicating that the Guide-it CRISPR Genome-Wide sgRNA Library System allows for sgRNA representation to be maintained throughout transduction and selection of the target cell population.

Optimized lentiviral sgRNA library vector and sgRNA scaffold design for high editing efficiency

The design for our sgRNA library vector, pLVXS-sgRNA-mCherry-hyg, has been optimized for performance and safety (Figure 2, Panel A). The self-inactivating lentiviral backbones used to deliver Cas9 and the sgRNAs provide puromycin and hygromycin selection markers, respectively, for isolation of transduced cell populations. In addition, the sgRNA library vector also expresses an mCherry reporter for rapid titer determination and visual confirmation of transduced cells. The inclusion of mCherry in the sgRNA vector also makes it easy to determine the optimal multiplicity-of-infection (MOI) required for transduction of the Cas9+ cell line with the sgRNA library (Figure 3). This is important, because ideally each Cas9+ cell should be transduced with only one sgRNA.

The sgRNA scaffold encoded by pLVXS-sgRNA-mCherry-hyg uses an optimized sequence for improved editing efficiency (Figure 2, Panel B). This modified scaffold maximizes the binding affinity between the sgRNA and the Cas9 endonuclease (Chen et al. 2013). Both the Cas9 and the sgRNA vectors are provided premixed with our Lenti-X Packaging Single Shots and Xfect Transfection Reagent. This mixture is lyophilized to produce a formulation that, with only the addition of water, is immediately ready for transfection of the included Lenti-X 293T producer cell line.

Optimized sgRNA scaffold design

Figure 2. Vector and sgRNA scaffold design used in the Guide-it CRISPR sgRNA library. Panel A. pLVXS-EF1a-Cas9-PGK-Puro and pLVXS-sgRNA-mCherry-hyg vector 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, derived from the Brunello library, are expressed from the human U6 promoter and use an optimized scaffold sequence for better Cas9 loading and editing efficiency (Panel B).

mCherry reporter

Figure 3. Determination of transduction efficiency of the sgRNA library lentivirus using mCherry fluorescence. sgRNA Library-containing lentivirus was produced following the instructions in the user manual: water was added to a tube of sgRNA Library Transfection Mix, vortexed, incubated for 10 min and then added to the provided Lenti-X 293T cells. Lentivirus was harvested after 48 hours and used to transduce Cas9-expressing A375 cells at varying MOIs. Transduced cells were plated and analyzed for transduction efficiency by fluorescence microscopy after 48 hours.

Results  

Editing activity of the Guide-it CRISPR Genome-Wide sgRNA Library in randomly selected clones

The editing activity of our sgRNA library system was tested by analyzing twenty randomly selected clones from a melanoma cell line (A375) transduced with both Cas9 and the sgRNA library. The sgRNA sequence was amplified from each clone, and the amplicon was sequenced to identify the sgRNA and design a primer set to amplify the sequence of the sgRNA's genomic target. The workflow schematic is shown in Figure 4, Panel A. Editing activity was then assessed using the Guide-it Mutation Detection Kit. The results of this resolvase-based assay are shown in Figure 4, Panel B. Cleavage products were observed for all isolated clones indicating a successful editing event. Each sgRNA found in the clones was also mapped to a correlation plot to determine the relative read counts of the cloned sgRNA within the original plasmid sgRNA pool and transduced cell population (Figure 4, Panel C). The even distribution of the randomly selected sgRNAs within the population suggests that there is minimal bias based on the starting representation level of the sgRNA. To further assess the editing activity in these clones, the amplified target sequences were cloned using the Guide-it Indel Identification Kit for sequencing of the indels. The sequencing results for two representative clones from the tested population, shown in Figure 5, demonstrate typical patterns of indels produced during these types of knockout experiments.

Checking randomly selscted closes after sgRNA library screen

Figure 4. Determination of editing activity of the Guide-it CRISPR Genome-Wide sgRNA Library in randomly selected clones. Panel A. After transduction of Cas9+ A375 cells with the sgRNA library and hygromycin selection, twenty clones were selected randomly and expanded for analysis. Panel B. Genomic DNA was isolated from each clone and analyzed for activity using the Guide-it Mutation Detection Kit. The results of the resolvase assay for 8 representative clones are shown; NTC: non-transduced cells, (+) PCR products treated with Guide-it Resolvase, (–) untreated PCR products. Panel C. The sgRNA sequences from the twenty clones were mapped against the correlation plot of the plasmid library and transduced gDNA. The sgRNAs isolated from the randomly-selected clones are marked with orange dots within the correlation plot of all sgRNAs (blue dots).

Sequencing results from randomly seleted clone showing indel patters

Figure 5. Indel identification in edited A375 clones Sequencing data from Cas9+/sgRNA+ A375 cells are shown for two representative clones from the edited population, demonstrating typical indel patterns.

Using the sgRNA library to screen for 6-thioguanine resistance

The efficacy of our lentiviral sgRNA library system was tested by performing a screen for genes involved in the purine salvage pathway. The selective agent, 6-thioguanine (6-TG; a purine analog), is metabolized intracellularly into its active metabolite by the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene. These metabolites are incorporated into DNA resulting in activation of the DNA mismatch repair system, which subsequently leads to apoptosis. Therefore, cells in which HPRT is knocked out should be able to survive in the presence of 6-TG.

Cas9+ cells were transduced with the sgRNA library virus at a low MOI and selected on hygromycin to select for Cas9+/sgRNA+ cells. The surviving cells were expanded and split into two populations. One was grown in the presence of 6-TG, whereas the other was maintained without selection as a reference-control population. After selection in 6-TG was complete, cells from both populations were expanded, and genomic DNA from each population was purified and analyzed by NGS (Figure 6).

Schematic for 6-TG screen using Guide-it CRISPR sgRNA library

Figure 6. Workflow schematic for a 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. One Cas9+/sgRNA+ cell population was exposed to 6-TG, while the control population was expanded under normal culture conditions without exposure to 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.

NGS analysis of the genomic DNA isolated from the cell population selected on 6-TG indicated a substantial loss of sgRNA representation in this population relative to the control population (or relative original plasmid sgRNA library), and a corresponding overrepresentation of all four sgRNAs targeting the HPRT gene (enriched as much as 30,000-fold; Figure 7, Panel B inset). In addition, representation of these guides within the control population was maintained when compared to their representation in the original plasmid sgRNA library (Figure 7, Panel A). Similarly, four sgRNAs targeting another gene, NUDT5 were also found to be over-represented in the screen and were identified as playing a role in the purine synthesis pathway by indirectly providing an eventual substrate on which the protein encoded by HPRT can act (Figure 7, Panel A). These results are similar to those reported by the developers of the Brunello library (Doench et al. 2016).

Results from 6-TG screen performed using genome-wide sgRNA library

Figure 7. Identification and analysis of sgRNAs isolated from cells after a 6-TG screen. Panel A. sgRNA representation was compared between the original plasmid library population used to prepare lentivirus, and the resulting transduced cell populations (selected and non-selected). All four sgRNAs targeting HPRT (red dots) and NUDT5 (black dots), respectively, were enriched in the 6-TG-selected population (blue dots). Representation of these sgRNAs in the plasmid library (gray) and transduced, unselected population (orange) is also shown. Panel B. After selection, sgRNA representation (blue) was shifted in response to 6-TG. Individual sgRNAs that were enriched are highlighted within this correlation (red and orange dots). The inset shows fold-enrichment of the four sgRNAs targeting the HPRT gene as compared to the plasmid library.

Conclusions  

The system contains lyophilized lentiviral transfection mixes for high-titer production of both Cas9 and sgRNA lentivirus, making it easy to establish and perform highly effective knockout screens while maintaining guide representation. sgRNA representation in every lot of the Guide-it CRISPR Genome-Wide sgRNA Library System is optimized such that >90% of all the sgRNAs in the plasmid library are within a 10-fold distribution range, and this representation is well maintained when the library is transduced into cells.

The optimized vector and sgRNA scaffold design, as well as the highly active sgRNAs, ensure high editing activity in transduced cell populations. In our hands, no bias for higher transduction efficiency was observed for sgRNAs with higher representation within the plasmid library (and vice versa). The pooled-library screen with 6-TG performed using the Guide-it CRISPR Genome-Wide sgRNA Library System and the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit successfully identified known key components of the purine salvage pathway.

Methods  

sgRNA representation within the library and the transduced cell population

The Brunello-derived sgRNA library was cloned into the pLVXS-sgRNA-mCherry-hyg Vector and amplified. The plasmid DNA was harvested and sequenced on an Illumina® MiSeq® to determine sgRNA representation. The sgRNA-containing lentivirus was produced following the user manual. Briefly, water was added to the Guide-it Genome-Wide sgRNA Library Transfection Mix, vortexed, and added to Lenti-X 293T cells after a 10-min incubation. Lentivirus was harvested after 48 hrs and used to transduce Cas9-expressing A375 cells. Transduced cells were selected on hygromycin (500 µg/ml) for 9 days, at which time genomic DNA was harvested and processed for NGS library preparation. The library was then sequenced on an Illumina MiSeq instrument to determine the relative read counts of each integrated sgRNA relative to the starting plasmid DNA population.

Determination of optimal MOI for transduction with sgRNA library virus

The sgRNA Library-containing lentivirus was produced following the included protocol. Water was added to the Guide-it Genome-Wide sgRNA Library Transfection Mix, vortexed, and added to Lenti-X 293T cells after a 10-min incubation. Lentivirus was harvested after 48 hrs and used to transduce Cas9-expressing A375 cells at varying MOIs to achieve approximately 30–40% transduction efficiency. Transduced cells were plated and analyzed for transduction efficiency by fluorescence microscopy and FACS after 48 hrs.

Resolvase assay for determining editing efficiency in the library population

Genomic DNA was isolated from twenty randomly selected Cas9+/sgRNA+ clones, target sequences were amplified, and PCR products were analyzed for mismatches using the Guide-it Mutation Detection Kit following the kit's protocol.

6-TG assay

A375 cells were transduced to produce a Cas9+/sgRNA+ cell population following instructions provided in the Guide-it CRISPR Genome-Wide sgRNA Library System User Manual. The cells were expanded and split into two populations. One line was selected using 12 µg/ml of 6-TG for 14 days, while the other line was maintained without adding 6-TG. The surviving cells were then expanded, and genomic DNA from the cells was isolated and processed for NGS analysis. The sequencing data were analyzed to determine which guide RNAs had increased or decreased in frequency in response to 6-TG.

NGS analysis

After conducting an sgRNA library screen, cell populations were harvested, genomic DNA (gDNA) was isolated, and sgRNA sequences were amplified from the integrated proviruses contained in the genomic DNA using the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit. The amplicons were then sequenced on an Illumina MiSeq platform. After sequencing, reads were trimmed using cutadapt software and the sgRNA sequence (20 bp) was mapped to the reference library or controls and the fold-change in sgRNA frequency determined using CLC Genomics Workbench. Results were then analyzed with Excel to determine which sgRNAs have increased or decreased in frequency due to the selective pressure applied in the screen.

References

Chen, B. et al. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell 155, 1479–1491 (2013).

Doench, J.G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

Doench, J.G. Am I ready for CRISPR? A user's guide to genetic screens. Nat. Rev. Genet. 19, 67–80 (2018).

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The Guide-it CRISPR Genome-Wide sgRNA Library System includes all of the components necessary to perform five lentiviral-based, genome-wide CRISPR/Cas9 knockout screens in human cells. Provided with the kit are lentiviral vectors for expression of Cas9 and >76,000 sgRNAs targeting >19,000 human genes (four guides per gene) in lyophilized, ready-to-transfect formats that simplify the production of high-titer lentivirus. The sgRNA library included with this kit is based on the Brunello library (Doench et al. 2016), and incorporates design features intended to maximize on-target efficacy and minimize off-target effects. To ensure balanced guide representation, we analyze the sgRNA library by NGS to confirm that >90% of the included sgRNAs fall within a 10-fold distribution range. Enrichment or depletion of sgRNAs in screened cells can be analyzed by NGS with the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647).

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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

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 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
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

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

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).

632647 Guide-it™ CRISPR Genome-Wide sgRNA Library NGS Analysis Kit 10 Rxns USD $725.00

The Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit enables preparation of next-generation sequencing (NGS) libraries from cell populations screened using the Guide-it CRISPR Genome-Wide sgRNA Library System (Cat. # 632646), allowing for identification of sgRNA sequences and corresponding gene knockouts that have increased or decreased in frequency under the conditions of the screen. The kit provides all the components necessary to prepare ten sequencing libraries (20 PCRs), including a genomic DNA purification kit, primers, PCR enzyme and buffers, and a PCR cleanup kit.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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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

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 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
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

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

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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632647: Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit

632647: Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit
632651 Guide-it™ CRISPR Genome-Wide Library PCR Kit 20 Rxns USD $349.00

The Guide-it CRISPR Genome-Wide Library PCR Kit is designed for NGS library preparation from cell populations screened using the Guide-it CRISPR Genome-Wide sgRNA Library System (Cat. # 632646), and is also sold as part of the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647). The kit contains all the primers and reagents necessary for amplification of proviral sgRNA sequences from screened cell populations for analysis by NGS. This kit includes enough reagents for performing 20 PCRs.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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632651: Guide-it CRISPR Genome-Wide Library PCR Kit

632651: Guide-it CRISPR Genome-Wide Library PCR Kit
631443 Guide-it™ Mutation Detection Kit 100 Rxns USD $552.00

The Guide-it Mutation Detection Kit contains all the reagents needed for PCR-based identification of insertions or deletions generated during cellular non-homologous end joining (NHEJ) repair. The first step is the amplification of the putative target sequence directly from cells. This kit uses Terra PCR Direct Polymerase Mix and Buffer, so there is no need to extract 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 for cleavage by the Guide-it Resolvase. Sufficient material is provided for 100 amplification and cleavage reactions.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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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
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

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

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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631443: Guide-it Mutation Detection Kit

631443: Guide-it Mutation Detection Kit

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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, 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).

631444 Guide-it™ Indel Identification Kit 10 Rxns USD $508.00

The Guide-it Indel Identification Kit is used for characterization of insertions and deletions (indels) generated by gene editing tools, such as CRISPR/Cas9. This kit contains all of the components needed to amplify, clone, and prepare modified target sites for DNA sequence analysis. This kit uses Terra PCR Direct to amplify targets directly from crude genomic DNA extracts. The resulting pool of fragments, which may contain a variety of indels, are cloned into a prelinearized pUC19 vector using the In-Fusion cloning system. Colony PCR of individual clones using Terra PCR Direct followed by DNA sequencing allows indel characterization.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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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
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 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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631444: Guide-it Indel Identification Kit

631444: Guide-it Indel Identification Kit

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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, 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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