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Deletion of regulatory elements with all-in-one CRISPR-Cas9 vectors

Authors
  • Cebola, I
Publication Date
Nov 16, 2020
Source
UPCommons. Portal del coneixement obert de la UPC
License
Unknown

Abstract

Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning by Golden Gate, by adapting the primer design.

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