Abstract
Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. Thus, experimentally validating gRNA activity is still the gold standard in defining the best gRNAs for gene editing experiments. One such approach for experimentally selecting gRNAs with high activity is fluorescent surrogate reporter vectors. We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.
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Acknowledgments
This work is partially supported by the Lundbeck Foundation (R219–2016-1375, R173–2014-1105), the Danish Research Council for Independent Research (DFF–1337–00128), the Sapere Aude Young Research Talent Prize (DFF-1335–00763A), the Innovation Fund Denmark (BrainStem), and Aarhus University Strategic Grant (AU-iCRISPR). Y.L is also supported by the Guangdong Provincial Key Laboratory of Genome Read and Write (No. 2017B030301011).
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Lin, L., Luo, Y. (2019). Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check. In: Luo, Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9170-9_5
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DOI: https://doi.org/10.1007/978-1-4939-9170-9_5
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