Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has revolutionized the field of genome engineering, medicine, as well as biotechnology. CRISPR/Cas9 is a form of bacterial defense mechanism that can be used for editing genomes by targeting a 20-nucleotide sequence using a guide RNA and nuclease enzyme called Cas9 enzyme that cleaves target gene. Different protocols have been provided; however, the success of CRISPR/Cas9 experiments is challenging. Here, we aim to describe the use of lentiviral vectors (lentiCRISPRv2/lentiGuide-Puro) for CRISPR/Cas9 genome editing and to provide strategies for minimizing off-targets. Troubleshooting advice will be provided based on experimental evidence. These guidelines will enable researchers and those with limited CRISPR/Cas experience to perform gene editing successfully.
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Acknowledgments
We would like to acknowledge the members of the Department of Molecular Biology and Genetics, Bilkent University, and TUBITAK for funding (Project No: 117Z227).
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Sa’id Ameen, Z., Cakiroglu, E., Senturk, S., Ibrahhim, A.U., Ozsoz, M. (2021). CRISPR/Cas9 Gene Editing in Mammalian Cells Using LentiCRISPRv2/LentiGuide-Puro Vectors. In: Islam, M.T., Molla, K.A. (eds) CRISPR-Cas Methods. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1657-4_18
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DOI: https://doi.org/10.1007/978-1-0716-1657-4_18
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