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

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


Show Resources
You May Also Like
MAY 17, 2022 9:00 AM PDT
MAY 17, 2022 9:00 AM PDT
Date: May 17, 2022 Time: 9:00am (PDT), 12:00pm (EDT), 8:00pm (CEST) Gene therapeutics have great potential to treat many severe diseases in an unprecedented, targeted manner. The biopharmace...
JUN 28, 2022 7:00 AM PDT
JUN 28, 2022 7:00 AM PDT
Date: June 28, 2022 Time: 3:00pm (BST), 4:00pm (CET), 9:00am (CST), 7am (PST) Light-sheet microscopy is an extremely versatile imaging technique with a vast range of implementations that are...
JUN 21, 2022 6:00 AM PDT
JUN 21, 2022 6:00 AM PDT
Date: June 21, 2022 Time: 6:00am (PDT), 9:00am (EDT), 3:00pm (CEST) The global understanding and practice of medicine is currently undergoing a revolutionary change. This shift to precision...
MAR 16, 2022 8:00 AM PDT
C.E. CREDITS
MAR 16, 2022 8:00 AM PDT
Date: March 16, 2022 Time: 8:00am (PDT), 11:00am (EDT), 5:00pm (CET) Handling of potent and/or hazardous substances is commonplace in sev.....
OCT 11, 2022 8:00 AM PDT
C.E. CREDITS
OCT 11, 2022 8:00 AM PDT
Date: October 11, 2022 Time: 8:00am (PDT), 11:00pm (EDT), 5:00pm (CEST) Multiomic profiling of cell populations at single-cell resolution is revolutionizing scientists’ understanding o...
APR 28, 2022 8:00 AM PDT
APR 28, 2022 8:00 AM PDT
Date: April 28, 2022 Time: 8:00am (PDT), 11:00am (EDT), 5:00pm (CEST) Human pluripotent stem cells (PSCs) and their derivatives hold great potentials in...
Loading Comments...
Show Resources