Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.

The energy landscape for R-loop formation by the CRISPR-Cas Cascade complex.

Clustered regularly interspaced short palindromic repeats (CRISPR) sequences and CRISPR-associated (Cas) genes comprise CIRSPR-Cas effector complexes, which have revolutionized gene editing with their ability to target specific genomic loci using CRISPR RNA (crRNA) complementarity. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base pairing between crRNA and the DNA target strand, forming an R-loop structure. Full R-loop extension is a prerequisite for subsequent DNA cleavage. However, the recognition of unintended sequences with multiple mismatches has limited therapeutic applications and is still poorly understood on a mechanistic level. Here we set up ultrafast DNA unwinding experiments on the basis of plasmonic DNA origami nanorotors to study R-loop formation by the Cascade effector complex in real time, close to base-pair resolution. We resolve a weak global downhill bias of the forming R-loop, followed by a steep uphill bias for the final base pairs. We also show that the energy landscape is modulated by base flips and mismatches. These findings suggest that Cascade-mediated R-loop formation occurs on short timescales in submillisecond single base-pair steps, but on longer timescales in six base-pair intermediate steps, in agreement with the structural periodicity of the crRNA-DNA hybrid.

Dynamic interplay between target search and recognition for a Type I CRISPR-Cas system.

CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors have to interrogate the entire genome for the presence of a matching sequence. Here we dissect the target search and recognition process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the effect of DNA supercoiling on the target recognition probability and demonstrate that Cascade uses facilitated diffusion for its target search. We show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

Purified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction.

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.

Characterizing microfluidic approaches for a fast and efficient reagent exchange in single-molecule studies.

Single-molecule experiments usually take place in flow cells. This experimental approach is essential for experiments requiring a liquid environment, but is also useful to allow the exchange of reagents before or during measurements. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins. Home-fabricated flow cells using two glass coverslips and a gasket made of paraffin wax are a widespread approach. The volume of the flow cell can be controlled by modifying the dimensions of the channel while the reagents are introduced using a syringe pump. In this system, high flow rates disturb the biological system, whereas lower flow rates lead to the generation of a reagent gradient in the flow cell. For very precise measurements it is thus desirable to have a very fast exchange of reagents with minimal diffusion. We propose the implementation of multistream laminar microfluidic cells with two inlets and one outlet, which achieve a minimum fluid switching time of 0.25 s. We additionally define a phenomenological expression to predict the boundary switching time for a particular flow cell cross section. Finally, we study the potential applicability of the platform to study kinetics at the single molecule level.

ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements.

Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. Previously, we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks (Fisher et al., 2017). However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualise ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerisation with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.

Structural Basis of Glycogen Biosynthesis Regulation in Bacteria.

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed ß helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.