Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture.

Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2Mcap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2Mcap for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. SCHEMA-based domain shuffling was used to map the specificity determining regions of Spl proteases, leading to a chimeric scaffold that supports specificity switching via subdomain exchange. The ability of A2Mcap to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future.

Error-Free Synthetic DNA by Molecular Dictation.

Synthetic DNA is the linchpin of the rapidly accelerating biotechnological era and is perhaps the most promising candidate for long-term digital data storage. Despite huge advances, manufacturing error-free DNA at low cost and high throughput remains challenging. Borrowing from well-established sequencing-by-synthesis technologies, we describe a new solution for DNA error correction.

UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution.

The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein's amino acids ('intra-gene epistasis'). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets.

Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology.

The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP 'hyperactivity' upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem ß-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner.

Insights into K-Ras 4B regulation by post-translational lysine acetylation.

Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.

Insights into Lysine Deacetylation of Natively Folded Substrate Proteins by Sirtuins.

Sirtuins are NAD(+)-dependent lysine deacylases, regulating a variety of cellular processes. The nuclear Sirt1, the cytosolic Sirt2, and the mitochondrial Sirt3 are robust deacetylases, whereas the other sirtuins have preferences for longer acyl chains. Most previous studies investigated sirtuin-catalyzed deacylation on peptide substrates only. We used the genetic code expansion concept to produce natively folded, site-specific, and lysine-acetylated Sirt1-3 substrate proteins, namely Ras-related nuclear, p53, PEPCK1, superoxide dismutase, cyclophilin D, and Hsp10, and analyzed the deacetylation reaction. Some acetylated proteins such as Ras-related nuclear, p53, and Hsp10 were robustly deacetylated by Sirt1-3. However, other reported sirtuin substrate proteins such as cyclophilin D, superoxide dismutase, and PEPCK1 were not deacetylated. Using a structural and functional approach, we describe the ability of Sirt1-3 to deacetylate two adjacent acetylated lysine residues. The dynamics of this process have implications for the lifetime of acetyl modifications on di-lysine acetylation sites and thus constitute a new mechanism for the regulation of proteins by acetylation. Our studies support that, besides the primary sequence context, the protein structure is a major determinant of sirtuin substrate specificity.

Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.

Re-epithelialization of cutaneous wounds in adult mammals takes days to complete and relies on numerous signalling cues and multiple overlapping cellular processes that take place both within the epidermis and in other participating tissues. Re-epithelialization of partial- or full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing. Live imaging after treatment with transgene-encoded or chemical inhibitors reveals that re-epithelializing keratinocytes repopulate wounds by TGF-ß- and integrin-dependent lamellipodial crawling at the leading edges of the epidermal tongue. In addition, re-epithelialization requires long-range epithelial rearrangements, involving radial intercalations, flattening and directed elongation of cells - processes that are dependent on Rho kinase, JNK and, to some extent, planar cell polarity within the epidermis. These rearrangements lead to a massive recruitment of keratinocytes from the adjacent epidermis and make re-epithelialization independent of keratinocyte proliferation and the mitogenic effect of FGF signalling, which are only required after wound closure, allowing the epidermis outside the wound to re-establish its normal thickness. Together, these results demonstrate that the adult zebrafish is a valuable in vivo model for studying and visualizing the processes involved in cutaneous wound closure, facilitating the dissection of direct from indirect and motogenic from mitogenic effects of genes and molecules affecting wound re-epithelialization.

Structural and Mechanistic Insights into the Regulation of the Fundamental Rho Regulator RhoGDIα by Lysine Acetylation.

Rho proteins are small GTP/GDP-binding proteins primarily involved in cytoskeleton regulation. Their GTP/GDP cycle is often tightly connected to a membrane/cytosol cycle regulated by the Rho guanine nucleotide dissociation inhibitor α (RhoGDIα). RhoGDIα has been regarded as a housekeeping regulator essential to control homeostasis of Rho proteins. Recent proteomic screens showed that RhoGDIα is extensively lysine-acetylated. Here, we present the first comprehensive structural and mechanistic study to show how RhoGDIα function is regulated by lysine acetylation. We discover that lysine acetylation impairs Rho protein binding and increases guanine nucleotide exchange factor-catalyzed nucleotide exchange on RhoA, these two functions being prerequisites to constitute a bona fide GDI displacement factor. RhoGDIα acetylation interferes with Rho signaling, resulting in alteration of cellular filamentous actin. Finally, we discover that RhoGDIα is endogenously acetylated in mammalian cells, and we identify CBP, p300, and pCAF as RhoGDIα-acetyltransferases and Sirt2 and HDAC6 as specific deacetylases, showing the biological significance of this post-translational modification.

Lysine-acetylation as a fundamental regulator of Ran function: Implications for signaling of proteins of the Ras-superfamily.

The small GTP-binding protein Ran is involved in the regulation of essential cellular processes in interphase but also in mitotic cells: Ran controls the nucleocytoplasmic transport of proteins and RNA, it regulates mitotic spindle formation and nuclear envelope assembly. Deregulations in Ran dependent processes were implicated in the development of severe diseases such as cancer and neurodegenerative disorders. To understand how Ran-function is regulated is therefore of highest importance. Recently, several lysine-acetylation sites in Ran were identified by quantitative mass-spectrometry, some being located in highly important regions such as the P-loop, switch I, switch II and the G5/SAK motif. We recently reported that lysine-acetylation regulates nearly all aspects of Ran-function such as RCC1 catalyzed nucleotide exchange, intrinsic nucleotide hydrolysis, its interaction with NTF2 and the formation of import- and export-complexes. As a hint for its biological importance, we identified Ran-specific lysine-deacetylases (KDACs) and -acetyltransferases (KATs). Also for other small GTPases such as Ras, Rho, Cdc42, and for many effectors and regulators thereof, lysine-acetylation sites were discovered. However, the functional impact of lysine-acetylation as a regulator of protein function has only been marginally investigated so far. We will discuss recent findings of lysine-acetylation as a novel modification to regulate Ras-protein signaling.

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation.

Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.

Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function.

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDiaN) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDiaN with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDiaN. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDiaN. We present a model of a mechanistically unexplored and new aspect of mDiaN regulation by liprin-α3.

p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium.

Adult zebrafish as a model system for cutaneous wound-healing research.

Upon injury, the skin must quickly regenerate to regain its barrier function. In mammals, wound healing is rapid and scar free during embryogenesis, whereas in adults it involves multiple steps including blood clotting, inflammation, re-epithelialization, vascularization, and granulation tissue formation and maturation, resulting in a scar. We have established a rapid and robust method to introduce full-thickness wounds onto the flank of adult zebrafish, and show that apart from external fibrin clot formation, all steps of adult mammalian wound repair also exist in zebrafish. Wound re-epithelialization is extremely rapid and initiates with no apparent lag phase, subsequently followed by the immigration of inflammatory cells and the formation of granulation tissue, consisting of macrophages, fibroblasts, blood vessels, and collagen. The granulation tissue later regresses, resulting in minimal scar formation. Studies after chemical treatment or with transgenic fish further suggest that wound re-epithelialization occurs independently of inflammation and fibroblast growth factor signaling, whereas both are essential for fibroblast recruitment and granulation tissue formation. Together, these results demonstrate that major steps and principles of cutaneous wound healing are conserved among adult mammals and adult zebrafish, making zebrafish a valuable model for studying vertebrate skin repair.