Microhomology-based choice of Cas9 nuclease target sites (original) (raw)
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- Published: 27 June 2014
Nature Methods volume 11, pages 705–706 (2014)Cite this article
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First we examined the mutations induced by ten TALENs and ten RGENs in human cells via deep sequencing (Supplementary Table 1 and Supplementary Methods). We focused our analysis on deletions because they are much more prevalent than insertions (98.7% vs. 1.3%, respectively, for TALENs; 75.1% vs. 24.9% for RGENs) and because microhomology is irrelevant for insertions. In aggregate, microhomologies of 2–8 bases were found in 44.3% and 52.7% of all deletions induced by TALENs and RGENs, respectively (Supplementary Fig. 1 and Supplementary Table 2). Thus, 43.7% (0.987 × 0.443) and 39.6% (0.751 × 0.527) of all the mutations induced by TALENs and RGENs, respectively, were associated with microhomology. At a given nuclease target site, the effect of these microhomology-associated deletions can be predicted. In the extreme cases, (i) all deletions cause frameshifts in a protein-coding gene or (ii) no deletions cause frameshifts. In contrast, one-third of microhomology-independent deletions result in in-frame mutations. Assuming that ∼60% of indels are microhomology independent on average, the fraction of in-frame mutations at a given site can range from 20% (60%/3 + 0%) to 60% (60%/3 + 40%), a threefold difference between the two extreme cases. Because most eukaryotic cells are diploid rather than haploid, the fraction of null cells carrying two out-of-frame mutations can range from 16% (0.40 × 0.40) to 64% (0.80 × 0.80), depending on the choice of target site.
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Figure 1: Prediction of nuclease-induced deletion patterns associated with microhomology.
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Acknowledgements
We thank J. Park for the help with website construction. This work was supported by the Plant Molecular Breeding Center of Next-Generation BioGreen 21 Program (PJ009081), the National Research Foundation of Korea (2013065262) and TJ Park Science Fellowship to S.B.
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- Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea
Sangsu Bae, Jiyeon Kweon, Heon Seok Kim & Jin-Soo Kim - Department of Chemistry, Seoul National University, Seoul, South Korea
Sangsu Bae, Jiyeon Kweon, Heon Seok Kim & Jin-Soo Kim
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S.B. and J.-S.K. have filed a patent application based on this work.
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Bae, S., Kweon, J., Kim, H. et al. Microhomology-based choice of Cas9 nuclease target sites.Nat Methods 11, 705–706 (2014). https://doi.org/10.1038/nmeth.3015
- Published: 27 June 2014
- Issue Date: July 2014
- DOI: https://doi.org/10.1038/nmeth.3015