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Abstract
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.
Highlights
Genome editing critically relies on selective recognition of target sites
Our results show that Cas[9] is less efficient than transcription activator-like effector nuclease (TALEN) in heterochromatin regions because Cas[9] tends to become encumbered by local searches on non-specific sites
In conclusion, we used a combination of single-molecule imaging and sequencing-based editing analysis to study the search dynamics of TALE and Cas[9] proteins in live cells
Summary
Genome editing critically relies on selective recognition of target sites. despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and transcription activator-like effector nuclease (TALEN) are programmable DNA search engines that query genomic sequences for target-specific editing[1]. Both Cas[9] and TALEN can recognize a custom genetic sequence but have strikingly different mechanisms of target-site binding[2]. In vitro single-molecule studies have shown that TALEs (nuclease-free analogs of TALENs) utilize a unique rotationally decoupled, “molecular zip-line” mechanism for target-site search along DNA; it does this by translating along the DNA backbone without rotating or tracking the major groove[6,7]. This combined strategy allows us to independently investigate the search mechanism as well as the editing efficiency of both genome-editing proteins
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