Abstract
CRISPR/Cas9 is a powerful genetic engineering technology that enables the introduction of genomic changes such as deletions and insertions of specific bits of DNA in cells with high precision. Compared to other programmable DNA nuclease such as ZFNs and TALENs, the specific binding of the Cas9 nuclease is mediated by a small guide RNA (gRNA), which can easily be designed to target any locus in the genome. The ease of generating novel gRNA vectors and its high efficiency has rapidly made CRISPR-Cas9 the dominant tool in gene editing applications, including gene knockout, knockin, tagging, etc. Here we describe our method for rapid and efficient generation of gene knockout or deletion cells using CRISPR/Cas9 within the time span of one month. The design of gRNAs, plasmid cloning, transfection, cell culturing, positive clone selection, and screening can be obtained from this method.
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Acknowledgments
The CRISPR projects are partially supported by grants from 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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Neldeborg, S., Lin, L., Stougaard, M., Luo, Y. (2019). Rapid and Efficient Gene Deletion by CRISPR/Cas9. 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_14
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DOI: https://doi.org/10.1007/978-1-4939-9170-9_14
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