Genome Editing
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Genome editing tip: A CRISPR RNA annealing step can increase editing efficiency

Improve your genome editing using the Alt-R® CRISPR System

Researchers performing genome editing experiments frequently ask us whether including a crRNA:tracrRNA annealing step is necessary to achieve the highest level of genome editing activity. So, we set up an experiment to test whether CRISPR RNA duplex formation functionally impacts genome editing.

Reagents optimized for genome editing—Alt-R® CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System provides the main components you need for successful genome editing. Based on the endogenous S. pyogenes CRISPR-Cas9 pathway, the high potency of the Alt-R CRISPR-Cas9 System is due to optimized Alt-R CRISPR crRNA and Alt-R CRISPR tracrRNA designs that increase editing efficiency. The system reagents and protocol provide:

  • Higher on-target potency than other CRISPR systems

  • Precision control with delivery of Cas9 ribonucleoprotein

  • Efficient delivery of the RNP with lipofection, electroporation, and microinjection

  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System and get protocols for hard-to-transfect cell lines at www.idtdna.com/CRISPR.

Functional test of RNA annealing step on genome editing

For this experiment we used Alt-R® CRISPR-Cas9 System RNAs (see the sidebar, Reagents optimized for CRISPR editing). For the annealing condition, Alt-R crRNAs targeting 2 HPRT sites (HPRT 38285 AS, HPRT 38509 S) were combined with Alt-R tracrRNAs in a 1:1 ratio in either IDTE (10 mM TRIS, pH 7.5; 0.1 mM EDTA) or Duplex Buffer (30 mM HEPES, pH 7.5; 100 mM Potassium Acetate), heated to 95ºC for 5 minutes, and allowed to slowly cool to room temperature. CRISPR RNAs treated with or without this annealing step were reverse transfected (Lipofectamine® RNAiMAX, Thermo Fisher Scientific) into Cas9 expressing HEK293 cells at 10, 20, or 30 nM final concentration. Genomic DNA was isolated 48 hr after transfection, and the HPRT target sites were PCR amplified and digested with T7EI (see the sidebar, T7El mismatch endonuclease assay for genome editing analysis). Resulting fragments were visualized on a Fragment Analyzer™ (Advanced Analytical Technologies).

How important is the crRNA:tracrRNA annealing step?

As seen in Figure 1, including the heating and cooling step to anneal CRISPR RNAs does improve genome editing activity at the lower transfected dose (10 nM). When this annealing step is omitted at this dose, there is a >30% loss in editing activity. 

In their paper, Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3'-terminal segment of guide RNA, Mekler et al provide an explanation for why omitting the crRNA:tracrRNA annealing step results in decreased genome editing activity. They show that the kinetics of RNP formation is ~9-fold slower when crRNA and tracrRNA are not preannealed [1, Fig 6].

While at higher transfection concentrations, the effect is less dramatic, the annealing step is still recommended to maximize genome editing efficiency; it can be particularly helpful for researchers working in sub-optimal or low efficiency settings.

CRISPR RNA annealing step improves genome editing by CRISPR RNA:Cas9 RNP

Figure 1. CRISPR RNA annealing step improves genome editing. Alt-R® crRNAs targeting 2 HPRT sites (HPRT 38285 AS, HPRT 38509 S; AS and S indicating anti-sense and sense strands, respectively) were mixed with tracrRNAs in a 1:1 ratio in either IDTE (pH 7.5) or Duplex Buffer and heated at 95ºC for 5 min, followed by slow cooling to room temperature. CRISPR RNAs treated with or without the heating and cooling step were reverse transfected with Lipofectamine® RNAiMAX (Thermo Fisher Scientific) into a stable HEK293-Cas9 expressing cell line at 10, 20, or 30 nM final concentration. Genomic DNA was isolated 48 hr after transfection, HPRT target sites were PCR amplified, and digested with 2U T7EI. Resulting fragments were visualized on a Fragment Analyzer™ (Advanced Analytic Technologies). Including a CRISPR RNA annealing step resulted in significantly improved genome editing at both target sites for 10 nM transfections. While this improvement was less dramatic at higher transfection concentrations, IDT scientists still recommend including this step to maximize genome editing efficiency.

We recommend that users titrate the amount of annealed CRISPR RNA that is transfected into their cells and use the lowest amount in the plateau phase of the dose-response curve (when increasing the amount delivered no longer improves genome editing efficiency) for ongoing experiments. This titration will ensure optimal editing while reducing potential off-target effects and should be done for each new cell line used.

Tested reagents and protocols for optimal genome editing

IDT scientists continue to develop improvements in CRISPR technology. We have demonstrated that you can further optimize genome editing by complexing CRISPR RNAs and Cas9 protein as a ribonucleoprotein (RNP) complex prior to transfection. See the data in the article, Improve your genome editing with the Alt-R® S.p. Cas9 Nuclease 3NLS and modified crRNAs. You can also obtain protocols for generating the RNP and deliveito cell lines at www.idtdna.com/CRISPR.

For more genome editing tips, see the Additional reading sidebar, or visit our DECODED online newsletter.

Reference

  1. Mekler V, Minakhin L, et al. (2016) Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3'-terminal segment of guide RNA. Nucl Acids Res, 44(6):28372845. 

     

    T7EI mismatch endonuclease assay for genome editing analysis

    We currently recommend using T7 endonuclease I (T7EI) for CRISPR mutation detection because the method is simple and provides clean electrophoresis results. T7EI analysis is compatible with a broad range of PCR buffers and does not usually require prior purification of the PCR product. Note that T7EI activity is sensitive to DNA:enzyme ratio and incubation temperature and time [1]. T7EI is able to recognize insertions and deletions of ≥2 bases that are generated by non-homologous end-joining activity in CRISPR experiments [2]. Because T7EI does not recognize 1 bp indels, it underrepresents the total editing. For a protocol, see the Alt-R® CRISPR-Cas9 System User Guide.

    References

    1. Mean RJ, Pierides A, et al. (2004) Modification of the enzyme mismatch cleavage method using T7 endonuclease I and silver staining. Biotechniques, 36(5):758–760.

    2. Vouillot L, Thélie A, Pollet N. (2015) Comparison of T7EI and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3: Genes|Genomes|Genetics, 5(3):407–415.

    Product focus—genome editing with Alt-R® CRISPR Reagents

    Alt-R CRISPR-Cas9 System

    The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

    • Higher on-target potency than other CRISPR systems
    • Precision control with delivery of Cas9 ribonucleoprotein (RNP)
    • Efficient delivery of the RNP with lipofection or electroporation
    • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

    Learn more about the Alt-R CRISPR-Cas9 System.


    Alt-R CRISPR-Cpf1 System

    The Alt-R CRISPR-Cpf1 System allows for new CRISPR target sites that are not available with the CRISPR-Cas9 System, and produces a staggered cut with a 5′ overhang. These reagents:

    • Enable genome editing in organisms with AT-rich genomes
    • Allow interrogation of additional genomic regions compared to Cas9
    • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
    • Permit efficient delivery of the RNP into cells by electroporation

    Learn more about the Alt-R CRISPR-Cpf1 System.


    CRISPR support tools

    Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.

    Find out more about IDT’s entire line of CRISPR products.

    Additional reading

    6 pieces of data that will change how you set up your CRISPR-Cas9 experiments—To improve the efficiency of CRISPR-Cas9 genome editing, IDT scientists evaluated several factors that influence how we design and perform genome editing experiments.Factors examined included Cas9 delivery, crRNA and tracrRNA length, which gRNA formats provide the most efficient on-target editing and elicit the least toxicity, the importance of protospacer size and site selection. Review the data and results for 6 important factors that were addressed. These experimental findings resulted in a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.

    Webinar: Alt-R® CRISPR-Cas9 System ribonucleoprotein delivery optimization—Genome editing using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) provides excellent editing efficiency, while reducing off-target editing and cell death. Watch this recorded presentation to find out how to easily generate and deliver CRISPR RNAs and Cas9 nuclease in an RNP format, using the optimized Alt-R CRISPR-Cas9 System.

    Genome editing: How stable is my CRISPR RNA:Cas9 RNP complex?—You can safely complex CRISPR RNAs with Cas9 in advance of your experiments and store these RNPs for future use. Store CRISPR RNPs at 4ºC for up to 2 weeks, or at –80ºC long-term. RNP complexes stored in this way provide the same high level of genome editing as freshly complexed RNPs.

    Improve your genome editing with the Alt-R® S.p. Cas9 Nuclease 3NLS and modified crRNAs—Product Spotlight: Methods for CRISPR-Cas9 genome editing are diverse, but not all of them perform equally well. We now offer the Alt-R™ S.p. Cas9 Nuclease 3NLS that, when used in combination the optimized Alt-R CRISPR crRNA and tracrRNA, provides a highly effective editing solution that is also easy to use.

    Author: Mollie Schubert, MS, is a research scientist, and Ellen Prediger, PhD, is a senior scientific writer, both at IDT.

    © 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


CRISPR-Cas9 Genome Editing

With Alt-R CRISPR-Cas9 System get the core components for improved on-target editing performance, while experiencing lower toxicity, and no innate cellular response.

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Related Content

Product page:

Alt-R CRISPR-Cas9 System

Webinar:

New RNA tools for optimized CRISPR/Cas9 genome editing

Learn how research conducted at IDT led to the development of a potent new set of CRISPR-Cas9 genome editing tools.

Poster:

Quantitative Measurement of CRISPR/Cas9 Gene Editing at the Level of Genomic DNA for sgRNA Site Selection Algorithm Development

User guide:

Alt-R CRISPR-Cas9 System User Guide