skip to main content

Title: Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
Abstract

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization byiterativesize-exclusion andrecombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-deadStreptococcus pyogenesCas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

Authors:
; ; ; ; ; ; ; ; ; ; ;
Publication Date:
NSF-PAR ID:
10305419
Journal Name:
Nature Communications
Volume:
12
Issue:
1
ISSN:
2041-1723
Publisher:
Nature Publishing Group
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform forglycosylationpathway assembly byrapidin vitromixing andexpression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by anN-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineeredEscherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.

  2. Abstract

    Domains are the structural, functional and evolutionary units of proteins. They combine to form multidomain proteins. The evolutionary history of this molecular combinatorics has been studied with phylogenomic methods. Here, we construct networks of domain organization and explore their evolution. A time series of networks revealed two ancient waves of structural novelty arising from ancient ‘p-loop’ and ‘winged helix’ domains and a massive ‘big bang’ of domain organization. The evolutionary recruitment of domains was highly modular, hierarchical and ongoing. Domain rearrangements elicited non-random and scale-free network structure. Comparative analyses of preferential attachment, randomness and modularity showed yin-and-yang complementary transition and biphasic patterns along the structural chronology. Remarkably, the evolving networks highlighted a central evolutionary role of cofactor-supporting structures of non-ribosomal peptide synthesis pathways, likely crucial to the early development of the genetic code. Some highly modular domains featured dual response regulation in two-component signal transduction systems with DNA-binding activity linked to transcriptional regulation of responses to environmental change. Interestingly, hub domains across the evolving networks shared the historical role of DNA binding and editing, an ancient protein function in molecular evolution. Our investigation unfolds historical source-sink patterns of evolutionary recruitment that further our understanding of protein architectures and functions.

  3. Abstract

    ArabidopsisVIRE2-INTERACTINGPROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsisvip2mutants are recalcitrant toAgrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly,AtVIP2overexpressor lines in Arabidopsis did not show an improvement inAgrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. In addition to regulating genes involved inAgrobacterium-mediated plant transformation,AtVIP2overexpressor line showed differential expression of genes involved in abiotic stresses. The majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated inAtVIP2overexpressor lines. Consistent with this observation,AtVIP2overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role inAgrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.

  4. Sloan Siegrist, M. (Ed.)
    ABSTRACT The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium -specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity tomore »the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth. IMPORTANCE Bacterial growth and division are extensively studied in model systems ( Escherichia coli , Bacillus subtilis , and Caulobacter crescentus ) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode—polar growth—is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae , in which many species, including Agrobacterium tumefaciens , exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.« less
  5. Abstract

    Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits.