DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.
Authors:
van Overbeek M, Capurso D, Carter MM, Thompson MS, Frias E, Russ C, Reece-Hoyes JS, Nye C, Gradia S, Vidal B, Zheng J, Hoffman GR, Fuller CK, May AP.
In:
Source:
Mol Cell
Publication Date:
(
2016
)
Issue:
63(4)
:
633-46
Research Area:
Basic Research
Molecular Biology
Platform:
Nucleofector® I/II/2b
4D-Nucleofector® 96-well Systems
Experiment
In order to determine the outcomes following Cas9 cleavage of double-stranded DNA cells were nucleofected with the complexes of Cas9 protein and sgRNA and DNA and the DNA repeir determined by amplicon sequencing. HEK cells were nucleofected using Nucleofector II shuttle platform and SF cell line kit as indictaed in the protocol. The experiment was repeated in other cells lines HCT116 (SE Cell Line nucleofection kit) and K562 (SF cell line neucleofection kit). They conclude that the DNA repair is non random and is unique to each target. To test the DNA repairr profiling in more phisiological conditions, HSCs were nucleofected with P3 Primary Cell Kit. The same profile of DNA repair was observed.
Abstract
The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.
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