Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

February 2020

Ida Höijer, Josefin Johansson, Sanna Gudmundsson, Chen-Shan Chin, Ignas Bunikis, Susana Häggqvist, Anastasia Emmanouilidou, Maria Wilbe, Marcel den Hoed, Marie-Louise Bondeson, Lars Feuk, Ulf Gyllensten, Adam Ameur

In this preprint, the authors propose a method for screening for off off-target CRISPR/Cas9 sites using PacBio and Oxford Nanopore sequencing. The amplification-free method purifies and sequences both on-target and off-target cut sites from a DNA sample. The method could prove useful for improving gRNA design and screening and suggests the ability to develop de Novo guides as opposed to requiring a reference genome. SageELF was used to fractionate (1-18KB) SMRTBells for PacBio Hi-Fi circular consensus sequencing.

Author Affiliations:
Uppsala University, Sweden
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA
Boston Children’s Hospital, Boston, MA
Monash University, Melbourne, Australia
DOI: 10.1101/2020.02.09.940486

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