Long-read sequencing reveals unforeseen CRISPR-Cas9 activity

Presented at: CRISPR 2020
C.E. Credits: P.A.C.E. CE Florida CE
Speaker
  • Associate Professor and Senior Bioinformatician, SciLifeLab, Uppsala University, Sweden
    Biography

      Adam Ameur is associate professor and senior bioinformatician at the SciLifeLab National Genomics Infrastructure in Sweden. His work is focused on technology development and novel sequencing applications for the study of human health and disease. Ongoing activities include the construction of a whole-genome reference dataset for genetic variation in the Swedish population, as well as introduction of long-read single molecule sequencing into clinical routine. Since 2017 he is also an adjunct researcher at Monash university in Melbourne, Australia.


    Abstract

    An extensively debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. We have developed a new protocol for detection of gRNA binding and Cas9 cleavage, based on amplification-free long-read sequencing. This method, which is named “off-target sequencing” (OTS), was assessed using a human cell line that was first re-sequenced using long and highly accurate reads to get a detailed view of all on- and off-target binding regions. The OTS method detected many Cas9 cleavage sites that were not reported by off-target prediction software, including in “dark” regions of the human genome inaccessible by standard short-read sequencing. We are currently investigating whether in vivo off-target editing can occur at these sites.

    Relevant publications:

    • Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity. Höijer I, Johansson J, Gudmundsson S, Chin CS, Bunikis I, Häggqvist S, Emmanouilidou A, Wilbe M, den Hoed M, Bondeson ML, Feuk L, Gyllensten U, Ameur A. bioRxiv 2020.02.09.940486; doi: https://doi.org/10.1101/2020.02.09.940486

    • Single-Molecule Sequencing: Towards Clinical Applications. Ameur A, Kloosterman WP, Hestand MS. Trends Biotechnol. 2019 Jan;37(1):72-85. https://www.ncbi.nlm.nih.gov/pubmed/30115375

    Learning Objectives:

    1. Introduction to long-read sequencing technologies

    2. New methods study CRISPR-Cas9 off-target activity


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