(338a) Target Search of Cas9 and Talen Is Dependent on Chromatin Architecture

The Discovery of gene editing proteins has revolutionized the biotechnology field. Now we have the capability to edit any gene in the mammalian genome that has numerous applications in therapeutics, agriculture, and disease prevention. Genome editing proteins need to find their target site accurately with minimal off-target effects. Despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. CRISPR/Cas9 and TALENs are programmable DNA search engines that query genomic sequences for target-specific editing (Figure A)¹. Both Cas9 and TALENs can recognize a custom genetic sequence but have strikingly different mechanisms of target-site binding². Cas9 can be programmed to find a specific DNA sequence upstream of a 3-nucleotide motif (protospacer adjacent motif or PAM) by designing a single guide RNA (sgRNA) that mediates target-site binding through DNA-RNA pairing³. The proximity of PAM site to the target site is necessary for Cas9 binding. On the other hand, the DNA-binding domain of a TALEN is comprised of a tandem array of 33-34 amino acid (aa)-long customizable monomers that can be assembled to recognize virtually any genetic sequence following a one-repeat-binds-one-base-pair recognition code^4,5. Target recognition code is embedded in the peptide backbone of the TALE protein, and, therefore, TALE proteins are designed specific to its target site.

Here, we use single-molecule imaging in live cells to directly study the behavior of Cas9 and TALEN⁶. Live-cell single-molecule fluorescence microscopy^7,8 was used to directly observe the search dynamics of TALE and Cas9 proteins in mammalian cells. The proteins are fused with a Halotag domain⁹ and were constructed using an in-house liquid handling robotic system¹⁰, enabling 1:1 stoichiometric labeling with JF 549 dye (Figure B)¹¹. Protein search dynamics were analyzed using two different imaging conditions: short-exposure times (10-20 ms) to study fast-moving protein diffusion kinetics and long-exposure times (500 ms) to characterize residence times of the bound protein molecules (Figure C). Both TALE and Cas9 exhibited two types of search behaviors, a “fast” diffusion and a “slow” diffusion with significant overlap. These results show that TALE and Cas9 are capable of switching (on a timescale of 20 ms) between these two diffusion modes.

To further understand the molecular origins of the slow-diffusing populations for TALE and Cas9, we analyzed individual trajectories of TALE and dCas9 to characterize search dynamics by calculating an instantaneous diffusion coefficient D_inst. The rapid 3-D search was distinguished from the local search using thresholds for D_inst. We observed that both TALE and dCas9 proteins transition rapidly between slow and fast D_inst ranges. TALE proteins transition between fast 3-D and slow local search along DNA in live cells, which is consistent with prior in vitro single-molecule studies of TALE proteins^4,5. Similarly, along with 3-D diffusion, dCas9 can also engage in local search of the genome in live cells. We also calculated the extent of local search by Cas9 and TALE. We found out that Cas9 spends more time (96 ± 1 ms) engaging in local DNA search than TALE (65 ± 0.3 ms) per local search cycle.

We next studied the search mechanism of TALE and dCas9 in the context of prominent genomic features in heterochromatin. We observed overall slower kinetics compared to euchromatin and differential search behavior depending on the chromatin context for both dCas9 and TALE. Jumping angle analyses demonstrate that both TALE and dCas9 encounter a considerably constricted search space as indicated by the highly skewed angular distribution in the heterochromatin region. TALE 16 (D = 2.35 µm²/s) showed significantly faster overall search dynamics compared to dCas9-gRNA9 (D = 1.93 µm²/s) in heterochromatin. Jumping angle distributions of TALE 16 are more uniformly distributed compared to that of Cas9-gRNA9, indicating that TALE can maneuver the tight heterochromatin environment more efficiently.

To assess the functional implications of differences in search behavior of TALE and dCas9 in heterochromatin, we constructed a series of TALENs and Cas9-gRNA variants capable of editing sequences present in highly repressed heterochromatin loci. In 11 out of 12 loci (91.66%), TALENs showed similar or higher editing activity in heterochromatin compared to Cas9 (Figure D). Together, the genome editing efficiency results are consistent with in vivo search dynamics results, showing that TALE proteins are more efficient than Cas9 in navigating dense heterochromatin regions of the genome due to enhanced ability to sample heterochromatin locally. Based on our results, we propose a mechanistic model for the search mechanisms of TALE and dCas9 in heterochromatin that explains the difference in their relative editing performance in euchromatin and heterochromatin (Figure E). TALE is able to find a target site embedded in mammalian heterochromatin with greater efficiency compared to dCas9. Overall, these results serve as a guide in selecting genome-editing proteins for the engineering of hard-to-edit heterochromatin regions of mammalian cells for general as well as therapeutic purposes.

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