Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check
| Autor: | Yonglun Luo, Lin Lin |
|---|---|
| Přispěvatelé: | Luo, Yonglun |
| Rok vydání: | 2019 |
| Předmět: |
Reporter vector
0303 health sciences Functional evaluation Computer science Cas9 In silico 030302 biochemistry & molecular biology Surrogate vector Computational biology C-Check Gene editing Green fluorescent protein 03 medical and health sciences chemistry.chemical_compound chemistry Genome editing CRISPR Guide RNA Gene DNA 030304 developmental biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493991693 Lin, L & Luo, Y 2019, Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System : C-Check . in Y Luo (ed.), CRISPR Gene Editing : Methods and Protocols . Springer Science+Business Media, Methods in Molecular Biology, vol. 1961, pp. 67-77 . https://doi.org/10.1007/978-1-4939-9170-9_5 |
| DOI: | 10.1007/978-1-4939-9170-9_5 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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