Tools for successful CRISPR/Cas9 genome editing

Use the Guide-it sgRNA In Vitro Transcription Kit to produce high yields of single guide RNAs (sgRNAs) for Cas9/CRISPR studies. The kit includes the Guide-it IVT RNA Clean-Up Kit, a phenol-free kit used to purify sgRNA following in vitro transcription (IVT) reactions. The purified sgRNA is ready for use in transfection, electroporation, or in vitro cleavage assays.

• Fast, efficient method for sgRNA production—produce any sgRNA in less than 3 hours
• Transcribe >4 µg of sgRNA per in vitro reaction
• Resulting sgRNAs can be used for in vitro screening and/or transfection

All necessary reagents are included in the kit:

• Scaffold Template
• In Vitro Transcription Buffer
• T7 Polymerase Mix
• RNase Free Water
• NucleoSpin Gel and PCR Clean-Up Kit
• High Yield PCR EcoDry Premix
• Guide-it IVT RNA Clean-Up Kit

Avoid delivering ineffective single guide RNAs (sgRNAs) to your cells—use the Guide-it sgRNA Screening Kit to test the efficacy of sgRNAs.

• Highly-optimized in vitro cleavage assay that allows estimation of cleavage efficiency
• Includes high-quality, recombinant Cas9 protein

All necessary reagents are included in the kit:

• Recombinant Cas9 Nuclease
• Reaction Buffer
• 10X BSA
• RNase Free Water
• 2 kb Control Fragment
• Control sgRNA
• NucleoSpin Gel and PCR Clean-Up Kit
• High Yield PCR EcoDry Premix

The Guide-it CRISPR/Cas9 systems provide a simple method to generate a vector that will deliver a target-specific sgRNA, Cas9 nuclease, and a bright fluorescent marker to your target cells.

• A single plasmid expresses all the components necessary for CRISPR/Cas9 targeting of your gene of interest
• The plasmid also expresses an exceptionally bright fluorescent protein (tdTomato or ZsGreen1) to monitor transfection efficiency and/or enrich for transfected cells by flow cytometry
• Plasmid construction is simple; design oligos specific to your target sequence, and clone into the pre-linearized plasmid using the included ligation reagents and high-efficiency competent cells

Use the Guide-it CRISPR/Cas9 Gesicle Production System to deliver Cas9 protein and a custom sgRNA to a broad range of cell types with very low cytotoxicity.

• Direct delivery of Cas9 protein instead of plasmid-based delivery prevents overexpression and genomic integration of Cas9, reducing off-target effects
• Tight control over the dose and timing of delivery
• The user makes gesicles containing active Cas9-sgRNA ribonucleoprotein (RNP) complexes from a producer cell line. Gesicles are harvested from the supernatant and added to the target cells

Use the Xfect RNA Transfection Reagent to efficiently transfect mammalian cells with messenger RNA (mRNA) and/or single guide RNA (sgRNA) with very low cytotoxicity.

• Transfection with Cas9 mRNA prevents genomic integration and persistent expression of Cas9, reducing off-target effects
• Simple, serum-compatible protocol
• Cotransfect with DNA using the Xfect Transfection Reagent

Electroporation of mammalian cells with Cas9-sgRNA ribonucleoprotein complexes allows you to minimize off-target effects while facilitating a fast turnaround for highly efficient gene-editing experiments. Try our Guide-it Recombinant Cas9 (Electroporation-Ready) for your CRISPR/Cas9-mediated gene editing experiments.

• The Cas9 protein solution is sterile and well tolerated by mammalian cells
• Minimizes off-target effects — better than plasmid-based delivery systems
• Works well in hard-to-transfect cell lines

The Guide-it Mutation Detection Kit provides a simple PCR-based technique to identify insertions and/or deletions generated in mammalian cells using CRISPR/Cas9 technology.

• Faster and more efficient than the Surveyor assay—amplify directly from your target cells
• Complete kit—Guide-it Resolvase, Terra PCR Direct Polymerase, and all buffers are included

Guide-it Mutation Detection Kit workflow.

All necessary reagents are included in the kit:

• Cell Extraction Buffers
• Guide-it Resolvase
• Terra PCR Direct Polymerase Mix
• Terra PCR Direct Buffer
• PCR-Grade Water

The Guide-it Genotype Confirmation Kit provides a simple method to determine if a given clone has mutations in one allele (monoallelic), both alleles (biallelic), or is unchanged (wild type). Expedite the laborious process of screening a large number of clones for those with the desired genotype.

• Streamlined protocol that uses direct amplification of target cellular genomic DNA
• Includes highly purified recombinant Cas9 nuclease for the in vitro cleavage reaction

The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety of indels created by CRISPR/Cas9 gene editing in a cell population.

• Kit includes components needed to amplify, clone, and prepare modified target sites for DNA sequence analysis
• Workflow has been optimized for fast results

One of the major challenges of knockin experiments is the production and delivery of the repair template along with the Cas9-sgRNA ribonucleoprotein complex. Although ssDNA repair templates have been shown to be more effective for gene editing than double-stranded DNA, their use is limited due to the difficulty of producing long templates or needing to use adeno-associated virus (AAV) for delivery. The Guide-it Long ssDNA Production System provides a fast and simple in vitro method for generating long ssDNA donor templates, which do not randomly integrate into the genome, are less toxic to cells, and do not require AAV production, resulting in a faster workflow and an improved gene editing efficiency.

• ssDNA does not randomly integrate into the genome, unlike dsDNA
• ssDNA does not express unless properly integrated into the genome, making identification of correctly edited cells more accurate when delivering expression cassettes
• ssDNA causes significantly less cytotoxicity in edited cells when compared with dsDNA

Guide-it Long ssDNA Production System workflow.

The Guide-it CRISPR Genome-Wide sgRNA Library System makes it easy to perform lentiviral-based, genome-wide phenotypic knockout screens using CRISPR/Cas9 in human cell lines. Included with the kit are expression systems for a genome-wide sgRNA library and Cas9 nuclease in our easy-to-use Lenti-X Single Shots format, enabling streamlined production of cell lines stably expressing both Cas9 and the sgRNA library.

• Pooled sgRNA library is validated to provide uniform representation of >76,000 sgRNAs targeting ~19,000 genes (four guides per gene)
• Optimized sgRNA library design (Brunello) maximizes on-target activity and minimizes off-target effects (Doench et al. 2016) while a modified sgRNA scaffold improves Cas9-sgRNA interaction
• Easy-to-use lyophilized Cas9 and sgRNA library transfection mixes; simply add water and apply to packaging cells

In addition to the library, we also offer a customized NGS analysis kit for our sgRNA library system so you can perform NGS analysis of your screens with ease. The Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit includes the necessary primers, reagents, and accessories to prepare an NGS-ready library from screened cell populations.

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).