Seek, but you may not find: TALEN edits heterochromatin region of the genome more efficiently than Cas9

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.
Seek, but you may not find: TALEN edits heterochromatin region of the genome more efficiently than Cas9

Gene editing proteins such as CRISPR/Cas9 and TALENs have revolutionized the field of biotechnology and opened myriads of avenues for new therapeutics, agricultural, and fundamental research applications. For designing gene-editing technologies of the future, it is essential to understand the efficiency and precision of editing. Gene editing proteins need to navigate the complex nuclear architecture and search for its specific target site among millions of possible sites. Single-molecule imaging technologies can track individual protein molecules in the nucleus and dissect their target search mechanisms. 

We employed single-molecule imaging of CRISPR/Cas9 and TALE proteins in the live-cell nucleus. We designed both CRISPR/Cas9 and TALE proteins targeting different regions of the genome. We hypothesized that protein structure encodes the nature of search mechanism and due to the inherent differences in CRISPR/Cas9 and TALEN protein structure, their search behavior should differ from each other. For instance, CRISPR/Cas9 requires unwiring the double helix to make specific contacts with the target site. In contrast, TALE protein wraps around the double helix and glides over DNA until it finds the target site upon which it shrinks along the pitch and binds tightly to the target site. By analyzing the diffusion characteristics of individual molecules, we showed that both CRISPR/Cas9 and TALE proteins are capable of performing a 3-D search as well as a more localized search in the genome but to different extents. Search mechanism has a direct impact on the specificity as well as the ability of these proteins to search for target sites embedded in specialized chromatin such as constitiutive heterochromatin or repetitive genomic regions. Gene editing proteins are sensitive to the genome packing. We observed that the duration of a protein engaginging local search determines its effectiveness in finding the target site in open chromatin but the same advantage becomes a detriment when the target site is located within a tightly packed chromatin region.

 Schematic of Cas9 and TALE proteins searching the nucleus. Cas9 (orange) gets trapped in tightly packed genome while TALE (blue) navigates the heterochromatin region of the genome more efficiently than Cas9.

 While Cas9 searched euchromatin effortlessly, surprising results came when we analyzed the genome editing proteins that targeted the heterochromatin region. TALEs navigated the heterochromatin region more efficiently, whereas Cas9 became stuck at nonspecific sites in the dense heterochromatin. We asked if the difference in the search behavior translated to functional differences. We assessed the editing efficiency of these proteins with custom-made TALE and CRISPR/Cas9 proteins targeting different sites in the heterochromatin. Out of the 12 heterochromatin sites, located in faculatitive or consititutive heterochromatin, TALEN showed higher or similar editing efficiency compared to Cas9. At the same time, Cas9 outperformed TALEN in editing the euchromatin regions of the genome. 

Our results have important implications for designing future specialized gene-editing applications that go beyond editing genomic regions in the open chromatin, such as targeting the silenced genome. We would also like to emphasize on the need for engineering current tools or discovering novel tools that can edit more efficiently in complex genomic loci in order to make the entire genome amenable to manipulation. We hope that our study will serve as a guideline in determining the structural and mechanistic features that such a genome-editing tool should have.

This article is based on: Jain, S., Shukla, S., et al. TALEN outperforms Cas9 in editing heterochromatin target sites. Nature Communications12, 606 (2021)

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