Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

A lonely electron blocks incoming pairs.

Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans. Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.

Rapid kinetics reveal surprising flavin chemistry in bifurcating electron transfer flavoprotein from Acidaminococcus fermentans.

Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. ß-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low-potential ferredoxin and the other to the high-potential α-FAD semiquinone (α-FAD•-). The resultant α-FAD hydroquinone (α-FADH-) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FAD•- to monitor formation of α-FAD•- or α-FADH-, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to ß-FAD at a rate of 920 s-1, yielding the charge-transfer complex NAD+:ß-FADH- with an absorbance maximum at 650 nm. ß-FADH- bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FAD•-. With α-FAD•-, the reduction of ß-FAD with NADH was 1500 times slower. Reduction of ß-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FAD•-. These data indicate that a nearby radical (14 Å apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf-Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.

Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii.

Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5'-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.

CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

Reduction of Flavodoxin by Electron Bifurcation and Sodium Ion-dependent Reoxidation by NAD+ Catalyzed by Ferredoxin-NAD+ Reductase (Rnf).

Electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) from Acidaminococcus fermentans catalyze the endergonic reduction of ferredoxin by NADH, which is also driven by the concomitant reduction of crotonyl-CoA by NADH, a process called electron bifurcation. Here we show that recombinant flavodoxin from A. fermentans produced in Escherichia coli can replace ferredoxin with almost equal efficiency. After complete reduction of the yellow quinone to the blue semiquinone, a second 1.4 times faster electron transfer affords the colorless hydroquinone. Mediated by a hydrogenase, protons reoxidize the fully reduced flavodoxin or ferredoxin to the semi-reduced species. In this hydrogen-generating system, both electron carriers act catalytically with apparent Km = 0.26 µm ferredoxin or 0.42 µm flavodoxin. Membrane preparations of A. fermentans contain a highly active ferredoxin/flavodoxin-NAD(+) reductase (Rnf) that catalyzes the irreversible reduction of flavodoxin by NADH to the blue semiquinone. Using flavodoxin hydroquinone or reduced ferredoxin obtained by electron bifurcation, Rnf can be measured in the forward direction, whereby one NADH is recycled, resulting in the simple equation: crotonyl-CoA + NADH + H(+) = butyryl-CoA + NAD(+) with Km = 1.4 µm ferredoxin or 2.0 µm flavodoxin. This reaction requires Na(+) (Km = 0.12 mm) or Li(+) (Km = 0.25 mm) for activity, indicating that Rnf acts as a Na(+) pump. The redox potential of the quinone/semiquinone couple of flavodoxin (Fld) is much higher than that of the semiquinone/hydroquinone couple. With free riboflavin, the opposite is the case. Based on this behavior, we refine our previous mechanism of electron bifurcation.

Comparative proteome analysis of Acidaminococcus intestini supports a relationship between outer membrane biogenesis in Negativicutes and Proteobacteria.

The presence of bona fide outer membranes in members of the class Negativicutes is anomalous as phylogenetic analyses place this class within the phylum Firmicutes. To explore the relationships of a representative member of Negativicutes, we have performed a whole proteome BLAST analysis of Acidaminococcus intestini, which indicates that a substantial proportion (7 %) of the A. intestini proteome is closely related to sequences from members of the phylum Proteobacteria. In addition, we have identified key proteins involved in outer membrane biogenesis in A. intestini. This work highlights the need for further studies to define the relationships and evolutionary history of the Negativicutes.

Modulations of the reduction potentials of flavin-based electron bifurcation complexes and semiquinone stabilities are key to control directional electron flow.

The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, ß-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces ß-FAD to ß-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of ß-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from ß-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.

Dehydration of (R)-2-hydroxyacyl-CoA to enoyl-CoA in the fermentation of alpha-amino acids by anaerobic bacteria.

Several clostridia and fusobacteria ferment alpha-amino acids via (R)-2-hydroxyacyl-CoA, which is dehydrated to enoyl-CoA by syn-elimination. This reaction is of great mechanistic interest, since the beta-hydrogen, to be eliminated as proton, is not activated (pK 40-50). A mechanism has been proposed, in which one high-energy electron acts as cofactor and transiently reduces the electrophilic thiol ester carbonyl to a nucleophilic ketyl radical anion. The 2-hydroxyacyl-CoA dehydratases are two-component systems composed of an extremely oxygen-sensitive component A, an activator, and component D, the actual dehydratase. Component A, a homodimer with one [4Fe-4S]cluster, transfers an electron to component D, a heterodimer with 1-2 [4Fe-4S]clusters and FMN, concomitant with hydrolysis of two ATP. From component D the electron is further transferred to the substrate, where it facilitates elimination of the hydroxyl group. In the resulting enoxyradical the beta-hydrogen is activated (pK14). After elimination the electron is handed-over to the next incoming substrate without further hydrolysis of ATP. The helix-cluster-helix architecture of component A forms an angle of 105 degrees, which probably opens to 180 degrees upon binding of ATP resembling an archer shooting arrows. Therefore we designated component A as 'Archerase'. Here, we describe 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans, Clostridium symbiosum and Fusobacterium nucleatum, 2-phenyllactate dehydratase from Clostridium sporogenes, 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, and lactyl-CoA dehydratase from Clostridium propionicum. A relative of the 2-hydroxyacyl-CoA dehydratases is benzoyl-CoA reductase from Thauera aromatica. Analogous but unrelated archerases are the iron proteins of nitrogenase and bacterial protochlorophyllide reductase. In anaerobic organisms, which do not oxidize 2-oxo acids, a second energy-driven electron transfer from NADH to ferredoxin, the electron donor of component A, has been established. The transfer is catalysed by a membrane-bound NADH-ferredoxin oxidoreductase driven by an electrochemical Na(+)-gradient. This enzyme is related to the Rnf proteins involved in Rhodobacter capsulatus nitrogen fixation.

Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells.

BACKGROUND: Cpf1 nucleases have recently been repurposed for site-specific genome modification. Two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium were shown to induce higher indel frequencies than SpCas9 when examining four randomly-selected target sequences for each type of nuclease. Whether they are a real match for Cas9 nucleases, however, remains to be verified. RESULTS: Here, we used AsCpf1 and LbCpf1 to induce homology directed repair, either single strand annealing (SSA) or homologous recombination (HR), in N2a mouse neuroblastoma cells. Exploiting a plasmid that contains two GFP halves with overlapping sequences and exploring 20 targets, on all but one both nucleases consistently performed with above 10 % efficiency. Several Cas9 nucleases have been previously characterised in order to find an orthogonal counterpart for the most widely used promiscuous SpCas9. Here, we found that AsCpf1 and LbCpf1 might be better candidates than three of the best such counterparts: Cas9 from Staphylococcus aureus, from Streptococcus thermophilus and from Neisseria meningitidis, when assessed for inducing efficient SSA mediated repair in N2a cells. When tested on genomic targets exploiting HR, both nucleases were able to induce the integration of a donor cassette with 1000 bp-long homologous arms. We also generated plasmids that express these Cpf1 nucleases together with their cognate crRNAs and that are equipped with type IIS restriction enzyme sites to facilitate spacer cloning. CONCLUSIONS: Our results suggest that employing As- or LbCpf1 nuclease to induce homology directed repair in N2a cells, although is less effective at present than employing SpCas9, it is an equally or more effective tool than the most frequently used orthogonal Cas9 counterparts of SpCas9. These findings support the position of Cpf1 nucleases on the side of SpCas9 on the palette of effective genome engineering tools. REVIEWERS: This article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.

Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii.

Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421-432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2 O2 , whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2 O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles' heel of anaerobes when exposed to air.