Invitrogen™ Precision gRNA Synthesis Kit

Catalog No.	Quantity
A29377	25 reactions

Catalog No. A29377 Supplier Invitrogen™ Supplier No. A29377

Description

Includes

BOX 1

• 5X TranscriptAidTM Reaction Buffer
• DNase I, RNase-free (1 U/μL)
• Nuclease-free water
• NTP mix (100 mM each of ATP, GTP, CTP,
• UTP in Tris buffer)
• Control gRNA forward and reverse primers (10 μM mix) 2

BOX 2

• Binding Buffer
• Wash Buffer 1 (concentrated)3
• Wash Buffer 2 (concentrated)4
• Nuclease-free water
• Gene JETTM RNA Purification Micro Column & Collection Tubes
• Elution Tubes, 1.5 mL

Simple, ready-to-use, all-in-one expression vector system for CRISPR-Cas9. Transfection-ready guide RNA (gRNA) in as little as four hours.

Simple, ready-to-use, all-in-one expression vector system for CRISPR-Cas9. GeneArt Precision gRNA Synthesis Kit is a complete system for rapid synthesis of guide RNA (gRNA) ready to complex with GeneArt Platinum Cas9 Nuclease or GeneArt CRISPR Nuclease mRNA. Starting with two short single-stranded oligos that code for the target sequence, the gRNA template is assembled with a T7 promoter in a short one-pot PCR reaction. The assembled product is then used as template in an in vitro transcription (IVT) reaction followed by a rapid purification step, yielding transfection-ready gRNA in as little as four hours. Resulting gRNA can also be co-transfected with our ready-to-transfect GeneArt Platinum Cas9 Nuclease or GeneArt CRISPR Nuclease mRNA. Both protein and mRNA Cas9 formats require no plasmid manipulation and so are amenable to high throughput and multiplex genome-wide cell engineering approaches.

Features of the GeneArt Precision gRNA Synthesis Kit include:

• Fast assembly and synthesis of any gRNA target in as little as four hours including template assembly
• High yield (>10 ug) and concentration (>200 ng/uL) of gRNA

For research use only. Not for use in diagnostic procedures.

Certifications

CoA

Recommended Storage

gRNA Prep Kit, store at -5 to -30°C and gRNA Cleanup Kit, store at room temperature

Specifications

• Concentration: 200 ng/μL
• Content And Storage: • gRNA Prep Kit, store at -5°C to -30°C
  • - gRNA Cleanup Kit, store at room temperature (For better long-term performance, store purification columns at 2°C to 8°C)
• Form: Liquid
• Format: Kit
• Reaction Speed: Fast
• Technique: CRISPR-Cas9
• Product Type: gRNA Synthesis Kit
• Promoter: T7
• Quantity: 25 reactions
• Shipping Condition: Approved for shipment at Room Temperature or on Wet or Dry Ice
• Sufficient For: 25 Reactions
• Yield: >10 μg at >200 ng/μL of gRNA
• Final Product Type: RNA
• No. of Reactions: 25 Reactions
• Starting Material: Oligos (DNA)

Frequently Asked Questions (FAQs)

I am having issues with low RNA yield using the Precision gRNA Synthesis Kit (Cat. No. A29377). What could be the cause and how can I improve my yield?

Low RNA yield can be due to several factors, including the pH of the binding buffer. Another common issue is RNA degradation. We recommend taking all precautions to prevent RNase activity. Some customers have reported an increase in yield by modifying the ethanol concentrations in the purification steps:
- 60% ethanol in the binding step (Step 2)
- 67% ethanol in Wash Buffer 1
- 80% ethanol in Wash Buffer 2
These modifications are detailed in the CellEvent Senescence Green Detection Kit (Pub. No. MAN0018221 A.0) .

If you continue to experience issues, please contact technical support at technicalsupport@thermofisher.com.

How do I check for off-target effects in my CRISPR-modified cell lines?

The only complete way to confirm that there are no off-target effects is to sequence the entire genome of your cell. Alternatively, a less thorough means of checking for off-target editing is to perform targeted sequencing of sequences with the highest probability of off-target effects (i.e., most similar to your CRISPR target region).

How many guide RNAs do you recommend designing against my desired edit locus?

A single guide RNA (gRNA) is all that is required for targeting, but we do recommend testing 2-3 gRNAs against each locus being targeted for cleavage. Testing multiple gRNAs increases the chances of finding a gRNA with high editing efficiency, which will reduce the screening time required to identify the clone of interest.

How does the Lipofectamine CRISPRMAX Reagent work?

The Lipofectamine CRISPRMAX Reagent combined with the proprietary enhancement properties of the Lipofectamine Cas9 Plus Reagent leads to efficient complex formation with the Cas9-gRNA ribonucleoprotein, for best delivery to the nucleus, helping to ensure high gene editing frequency for a wide range of cell types.

Can the Lipofectamine CRISPRMAX Reagent be used to deliver proteins other than the Cas9 nuclease?

Although the Lipofectamine CRISPRMAX Reagent was developed for the delivery of the Cas9-gRNA ribonucleoprotein, there is potential for other protein complex applications.

Can the Lipofectamine CRISPRMAX Reagent also be used to deliver the donor plasmid in addition to the Cas9 nuclease protein and gRNA?

The Lipofectamine CRISPRMAX Reagent was developed to efficiently deliver the Cas9-gRNA ribonucleoprotein complex. We recommend that you first deliver the donor plasmid with Lipofectamine 3000, then follow with delivering the Cas9-gRNA complex with Lipofectamine CRISPRMAX Reagent for best editing efficiency.

What gRNA controls do you recommend?

The Precision gRNA Synthesis Kit (Cat. No. A29377) includes primers for synthesis of gRNA targeting safe harbor locus HPRT. We also have a fully processed, fully validated HPRT gRNA with GCD primers for confirmation of cleavage available from our custom services team.

What in vitro transcription and/or cleanup kits should I purchase to go along with the GeneArt Precision gRNA synthesis kits?

No other kits are required for this process. All necessary components are included in the kit.

For Research Use Only. Not for use in diagnostic procedures.