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Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector

Part of the Methods in Molecular Biology book series (MIMB,volume 2162)


Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.

Key words

  • CRISPR/Cas9
  • Genome editing
  • IDLV
  • Off-target activity

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  • DOI: 10.1007/978-1-0716-0687-2_14
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This work is supported by a grant RB3-02161 from California Institute of Regenerative Medicine (J.K.Y.) and a grant from Nesvig Foundation.

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Correspondence to Jiing-Kuan Yee .

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Wang, X., Wu, Y., Yee, JK. (2021). Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector. In: Fulga, T.A., Knapp, D.J.H.F., Ferry, Q.R.V. (eds) CRISPR Guide RNA Design. Methods in Molecular Biology, vol 2162. Humana, New York, NY.

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  • Print ISBN: 978-1-0716-0686-5

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