Arg917 pdf

Readers are welcome to comment on the online version of the aper. All data to support the conclusions can be found in the figures and the source data file. More information is summarized in Supplementary Table 1. All the plasmids and oligo sequences used in this study are summarized in Supplementary Table 2. Biochemical and in vivo data demonstrate that CasX is active for E.

These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a. In addition to their microbial functions, RNA-guided DNA binding and cutting have proven to be transformative tools for genome and epigenome editing across wide-ranging cell types and organisms 3 — 5.

However, without full reconstitution of the CasX enzyme, it was not possible to determine the basis of the previously reported plasmid interference activity. We further find that CasX induces programmable, site-specific genome repression in E. Eight molecular models of CasX in different states Supplementary Table 1 , determined by cryo-electron microscopy cryo-EM , reveal an unanticipated quaternary structure in which the RNA scaffold dominates the architecture and organization of the enzyme.

Distinct conformational states observed for CasX suggest an ordered non-target and target strand cleavage mechanism that may explain how other CRISPR-Cas enzymes with a single active site, such as Cas12a, achieve double-stranded DNA cleavage 5 , 9 , To determine the molecular function, we undertook biochemical studies of the wild-type CasX from Deltaproteobacteria DpbCasX.

Cartoons are scaled according to the gene size. TS and NTS indicate the target strand and non-target strand, respectively. P indicates the cleavage product. The cleavage fraction is calculated based on the NTS band density compared to input NTS band density at reaction time of 0 min. Lane M shows labeled ladders.

The cleavage fraction is calculated based on the trans-ssDNA band density compared to input trans-ssDNA band density at reaction time of 0 min. We found that trans-ssDNA cutting activity was minimal compared to that observed for LbCas12a or for another related enzyme Cas12b Fig.

These results indicate that the presence of a single active site for double-stranded DNA cleavage does not necessarily correspond to target-dependent trans-cleavage activity, raising the possibility of structural or mechanistic differences between these enzyme families.

We found that DpbCasX reduced cell viability in a genome cleavage assay, at close to but slightly less than SpyCas9 activity levels Fig.

Surprisingly, we found that mutation of all three DpbCasX residues was required for maximal gene repression activity Extended Data Fig.

This assay has been repeated more than 3 times with consistent results. We next tested whether CasX is capable of inducing cleavage and gene editing of mammalian genomes. Using a previously reported destabilized-GFP disruption assay 15 Fig. Since the guide RNA recognizing the coding strand functioned more robustly in each case, we wondered if the additional GFP gene disruption observed for guide 3 could be explained by RNA targeting 4 , However, there was no recovery of GFP expression in these cells over time Fig.

Next, we explored the effect of CasX-sgRNA-encoding plasmid concentration on the extent of genome editing. Sub-cloning PCR-amplified segments of the GFP locus from treated cells revealed a wide variety of indels, many of which map to the cut sites identified in vitro Extended Data Fig.

Finally, we compared the editing efficiency of PlmCasX to that of SpyCas9, which has been optimized over the past 6 years for high efficiency. These results demonstrate that CasX belongs to a third distinct class of CRISPR system capable of targeted genomic regulation and editing, and motivated experiments aimed at determining the structural and mechanistic basis for these activities. Three-dimensional particle classification and refinement revealed two conformational populations of the ternary complex at resolutions of 3.

These two conformational states were also observed by cryo-EM analysis of a CasX complex containing a full R-loop 45 bp DNA substrate and refined at resolutions of 3. We discuss this domain and its function in depth in the section below.

Instead, we postulate that the TSL domain is responsible for target strand placement in the RuvC active site In other enzymes, similar loops or hairpin elements containing a large hydrophobic amino acid tyrosine or phenylalanine are thought to be involved in DNA strand separation 22 — Different domains are colored as in a. Residues Arg and Gln are shown as red sticks. The TSL-loop is shown as a red ribbon.

The positions of the RuvC active site residues are shown as red sticks to illustrate the distance to the active site from the TSL domain elements. The right panels show a zoomed-in views of the TSL domain. Protein residues interacting with gRNA recognition are shown as magenta sticks. CasX is shown as a transparent grey cartoon, and the residues responsible for cleavage activity are shown in red.

The nucleic acids are shown as ribbons to emphasize the rotation of the RNA-DNA duplex required for the transition between the two states. Mutation of the triplex or the scaffold stem diminished CasX activity in vivo, whereas truncated versions of the perpendicular stem loop retained activity Extended Data Fig. This preference suggests that non-target strand DNA is cleaved by the RuvC domain first, followed by displacement and target strand cleavage. The schematic of the DNA probe used for each data collection is shown on the left, with cleavage sites shown by arrowheads.

The RuvC domain is indicated in each map. All the EM maps are low-pass filtered to 4. The relative percentage of particles belonging to each state revealed by cryo-EM analysis is shown in the right panel. In our sequential model of CasX-mediated DNA cleavage, a substrate-bound complex mimicking the intermediate state that occurs after non-target strand cleavage should preferentially adopt State II.

This conformation is incompatible with double-stranded DNA cleavage at position 22 and is unlikely to occur natively Fig.

Although it showed similar physical behavior to that observed for the wild-type CasX on a size exclusion column Extended Data Fig. This finding also hints at the interesting possibility that the self-contained NTSB domain could be introduced into or acquired by other enzymes to assist with or stabilize double-stranded DNA binding. The reactions were analyzed at time points from 0 to minutes. Completely base-paired probe and a bubbled probe were used to test the on-target activity, and a random 50nt oligo was used to test the trans- cleavage activity.

These functional insights will enable the continued development of CasX as a third unique platform for RNA-programmed genome editing. The compact size, dominant RNA content, and minimal trans-cleavage activity of CasX differentiate this enzyme family from Cas9 and Cas12a, providing opportunities for therapeutic delivery and safety that may offer important advantages relative to existing genome editing technologies. Standard cloning techniques were used to create all plasmids. Plasmid construction and retention was ensured with AmpR and CmR as selectable markers They were grown on media containing two antibiotics to ensure selection for both plasmids.

Colonies were picked in triplicate from these plates into EZ-RDM liquid media and grown for 12 hours. The guide sequence was moved onto the protein-encoding plasmid and ng of this was used in the transformation. Two technical replicates of tenfold serial dilutions were spotted onto plates containing antibiotics for plasmid s used in the transformation. G essentially as described by Tiscornia et al. The next day cells were selected with 1.

Cells were passaged one time to maintain sub-confluent conditions. For extended assays cells were passaged and reanalyzed on the date notes. The PlmCasX vs SpyCas9 EGFP line 1 disruption assays were done as described above but the protocol was improved by using lipofectamine instead of , according to manufactures protocols, selecting with 1. T7EI assays were performed as previously described with slight modification Ligation products were transformed into cells and colonies were grown overnight before being picked, miniprepped, and Sanger sequenced at the UC Berkeley DNA Sequencing Facility.

Sequencing results were aligned to the target and visualized via Snapgene. The gene encoding CasX was sub-cloned into the 2CT expression vector. All the proteins were expressed using Rosetta E. The main culture was grown to an OD of 0. Cells were lysed by sonication and pelleted at g for 30 min. Protein was eluted from the heparin column using a sodium chloride gradient up to 1M sodium chloride.

For the wild type protein, there were two peaks containing CasX. The peak that eluted at lower salt concentration was found to contain inactive and aggregated protein and was not pooled; only the second peak contained active protein and only that protein was used for the assays. Pure protein was concentrated and flash-frozen. The overall expression yield was similar, but the amount of the well-folded protein second peak was lower than in case of wild type protein.

In vitro transcription template:. Resulting stocks of protein and sgRNA were mixed in molar ratio and incubated for 10 min at room temperature to produce active complex. Cleavage reactions were conducted in 1x reaction buffer; the radiolabeled probe concentration was 2 nM. In the cleavage assays used to determine the DNA cut sites Fig. All the nucleotide sequences and plasmids used in this study have been summarized in Supplementary Table 2.

Immediately after glow-discharging the grid for 14 seconds using a Solaris plasma cleaner, 3. Micrographs were recorded using SerialEM on a Gatan K2 Summit direct electron detector operated in super-resolution mode We collected a 4. For CasX binary and ternary complexes, the 28 frames we skipped the first 2 and last 2 frames of each image stack in super-resolution model were aligned, decimated, summed and dose-weighted using Motioncor2 CTF values of the summed-micrographs were determined using Gctf Initial particle picking to generate template images was performed using EMAN2 About 10, particles were selected and then imported into Relion2.

Particle picking for the complete dataset was carried out using Gautomatch by Kai Zhang, unpublished with templates generated in the previous 2D classification. Local CTF was re-calculated by Gctf with the determined box files. Particles were extracted from the dose-weighted, summed micrographs in Relion2.

For images obtained with a Volta Phase Plate, following preprocessing the CTF and phase-shift values of the summed-micrographs were determined using Gctf and then applied to dose-weighted, summed micrographs for further processing. The reactions were stopped by adding 50 mM Tris final concentration. Cross-linked samples were then digested by trypsin and purified for mass spectrometry analysis.

Cross-linked peptides were identified using an upgraded version of pLink In pLink, parameter of cross-linker was set to BS3. Parameter of enzyme was set to trypsin with up to three missed cleavages. Precursor mass tolerance and fragment mass tolerance were both set to 20 ppm. At least 6 amino acids were required for each peptide chain. Carbamidomethylation on cysteine was searched as a fixed modification. Oxidation on methionine was searched as a variable modification.

The protein main chain was manually traced in Coot After main chain building, side chains were assigned manually based on the EM map in Coot and then were further improved using the cryo-EM map of State I with the full R-loop at a resolution of 3. To improve backbone geometry, the atomic model was subjected to PHENIX real space refinement global minimization and ADP refinement with secondary structure, Ramachandran, rotamer, and nucleic-acid restraints.

The final model was validated using Molprobity 39 and cross-linking MS data. Additional DNA nucleotides were manually built in Coot. DNA nucleotides were manually modified in Coot.

Precipitated proteins were collected by centrifugation. The samples were excited with a 14keV X-ray beam and fluorescence spectrums were collected. Elements in the sample were identified based on characteristic emission energies. The family tree was visualized using iTOL v3 A multiple sequence alignment of the resulting RuvC domains was used to extract the percent identity of each pair of orthologs and generate a heatmap illustrating the pairwise comparisons of the RuvC domains.

Histograms of the frequency of occurrence of each identity value in pairwise comparisons of Cpf1, Cas9, and CasX were plotted from the heatmap. All the reagents used in this work are commercialized.

All the protocols have been described in detail above. All the software used in this work have been noted with references and available for academic usage.

Further information request should be addressed to J. Further information on research design is available in the Nature Reporting Summary linked to this paper. Triangle denotes collapsed branches.

Bootstrap values are indicated as percentage points; values above 88 are shown between the major branches. High identity is shown in blue with low identity shown in red. Histograms representing interfamily and intrafamily sequence identity value distributions are shown along the edge. Target regions for guide 1 to 9 are marked a long the gene.

The Cas proteins and guide RNAs used in each assay are marked. The representative S size exclusion traces by UV absorbance are shown. TS indicates the target strand from target DNA. All the reconstitutions have been repeated for more than 3 times with consistent results. By 2D based manual screening, , good particles were selected for 3D classification into 4 classes.

State I and State II were then independently refined to 3. Panels c and d are directly taken from the standard output of Cryosparc. Panels c and d are standard outputs of Cryosparc. Two orientations are presented for each model. The protein hit with highest Z-score for each target is shown in left panel. The hits are marked with protein name and PDB code. Z-score above 8 indicates a high degree of similarity. Z-score below 8 but above 2 indicates moderate similarity usually irrelevant random match.

Z-score below 2 indicates noise. Zinc ribbon residues are colored in blue. The primary sequence across TSL-loop is shown. Tyrosines are marked with teal circles. Positive charged residues are marked with red circles. Bovine erythrocyte carbonic anhydrase that contains zinc in the active site was used as a positive control. Representative Zinc peaks appeared in the purified CasX sample but not in the purified Cas9 sample. Protein parts are colored in cyan, and nucleic acid in dark gray.

Guide RNAs are subtracted out from the complexes and shown as ribbons in bottom panels, independently. Mass ratio of protein and guide RNA is shown in the right. Protein mass occupancies are colored in cyan, and guide RNA in dark gray. Sequence for the fused single guide RNA is shown. Cas9 is used for positive control. NC indicates the non-complementary CasX guide.

WT indicates the complementary wild type guide for CasX. The scale bar is 50nm. Representative reference-free 2D class-averages are shown on the bottom panels for the three samples. The scale bar is 20nm. The two lysine within a cross-linked pair are connected with purple curve.

State I and State II were then independently refined to 4. Panels c and d are directly adopted from the standard outputs of Cryosparc. All the assays have been repeated for 3 times with consistent results. The different protein domains are colored as in Figure 3. We thank D. Toso and P. Grob for expert electron microscopy assistance, and A. Chintangal and P.

Tobias for computational support. Savage, J. Cofsky, and A. Wright for comments on the manuscript. This project was funded by NSF grant no.

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