CRISPR gRNA Activity Validation in Precision Animal Breeding

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CRISPR gRNA Activity Validation in Precision Animal Breeding

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How efficiently CRISPR system works largely depends on the sgRNA. BioVenic provides gRNA activity detection services in order to address the uncertainty of candidate sgRNAs. Our services utilize a range of sgRNA screening technologies, which allows researchers to identify the most appropriate sgRNA before delivering genome editing tools to living animal cells.

Schematic Diagram of Factors Affecting gRNA Activity. (BioVenic Original) Fig.1 Schematic Diagram of the CRISPR Genome Editing System. (BioVenic Original)

Services of gRNA Activity Verification

Single-strand Annealing (SSA) Assay

Single-strand annealing (SSA) assay can verify the editing ability of gRNA editing vectors for naked DNA and is a common method for the preliminary evaluation of gRNA activity. The reporter plasmid in the SSA assay contains two non-active luciferase coding fragments. After efficient cleavage to form DNA double-strand breaks, the luciferase coding fragment will be repaired through homologous recombination using the SSA mechanism. BioVenic initially assesses the activity of the gRNA solely through fluorescence detection.

In Vitro Cleavage Assay

The editing efficiency of the CRISPR system is related to gRNA. A target gene has multiple editable targets. Different targets can be designed with corresponding gRNAs, and their cleavage activities may vary. Therefore, it is necessary to screen out the gRNA with the highest cleavage efficiency. BioVenic conducts in vitro testing to evaluate the editing activity of various gRNAs. By utilizing the Cas enzyme to cleave DNA sequences in vitro and measuring the percentage of gRNA-mediated DNA cleavage, we will readily assess the activity and efficiency of gRNAs for our customers.

In Vivo Cleavage Assay

BioVenic uses a mismatch endonuclease assay to detect the endogenous activity of gRNAs. Utilizing the functional characteristics of T7E1 endonuclease to identify and cleave mismatched hybrid DNA double strands, the design of primers on both sides of the target site is necessary for PCR amplification. We only need to estimate the ratio of enzyme-cleaved bands to un-cleaved bands in order to assess the endogenous activity of the gRNA.

Comparison of Different Activity Validation Services

Services	Contents	Features of the Assay
SSA Assay	1. Design and synthesis of gRNA target primers. 2. Construction of pSSA-luc vector. 3. Cell Transfection.	Simple design, easy operation and remarkable effect.
In vitro Cleavage Assay	1. In vitro transcription of gRNAs. 2. CRISPR/Cas in vitro enzyme digestion reaction.	Only target primer design is required, no cell platform support is required, and low cost.
In vivo Cleavage Assay	1. Genome extraction. 2. Target primer design. 3. T7E1 enzyme digestion reaction.	The screening cycle is short and closer to the situation during editing.

Why Choose Us?

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Multi-faceted one-stop gRNA activity validation.

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Sufficient pre-experimental screening and evaluation of gRNA.

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Reduce the impact of inefficient editing on project schedules.

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Comprehensive customization services for gRNAs in precise animal genome editing.

BioVenic relies on its de novo gene synthesis platform, comprehensive molecular biology laboratory, and high-throughput sequencing technology to rapidly validate the editing activity of gRNA in the CRISPR system for farmed animals. Through three evaluation techniques, the efficiency of editing is systematically and comprehensively verified at three different levels. By customizing your gRNA validation service, you can easily obtain the best editing tools and significantly advance the research progress of animal precision breeding projects. Please feel free to contact us for intuitive gRNA activity verification results.

Reference

Karmakar, Subhasis, et al. "In vitro Cas9 cleavage assay to check guide RNA efficiency." CRISPR-Cas Methods: Volume 2 (2021): 23-39.