TY - JOUR
T1 - TALEN outperforms Cas9 in editing heterochromatin target sites
AU - Jain, Surbhi
AU - Shukla, Saurabh
AU - Yang, Che
AU - Zhang, Meng
AU - Fatma, Zia
AU - Lingamaneni, Manasi
AU - Abesteh, Shireen
AU - Lane, Stephan Thomas
AU - Xiong, Xiong
AU - Wang, Yuchuan
AU - Schroeder, Charles M.
AU - Selvin, Paul R.
AU - Zhao, Huimin
N1 - Funding Information:
The authors thank L. Lavis for generously providing HaloTag ligands for imaging experiments and Tarun Chhabra and Neetesh Sharma for assistance with statistical data analysis. We also thank Kai Wen Teng and Duncan Nall from Selvin lab for their help with initial imaging experiments. We would also like to thank Guanhua Xun and Emily Gaither for help with TALEN synthesis. This work was supported by the National Institutes of Health (1U54DK107965 and 1UM1HG009402 to H.Z. and NS100019 to P.R.S.) and the National Science Foundation (PHY 1430124 to P.R.S.).
PY - 2021/12
Y1 - 2021/12
N2 - Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.
AB - Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.
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U2 - 10.1038/s41467-020-20672-5
DO - 10.1038/s41467-020-20672-5
M3 - Article
C2 - 33504770
SN - 2041-1723
VL - 12
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 606
ER -