RESUMO
The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/metabolismo , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/metabolismo , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/genética , Linhagem Celular Tumoral , DNA/química , DNA/genética , Evolução Molecular Direcionada/métodos , Humanos , Mutação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/genética , Streptococcus pyogenes/genética , Especificidade por SubstratoRESUMO
The accurate determination of protein-protein interactions has been an important focus of molecular biology toward which much progress has been made due to the continuous development of existing and new technologies. However, current methods can have limitations, including scale and restriction to high affinity interactions, limiting our understanding of a large subset of these interactions. Here, we describe a modified bacterial-hybrid assay that employs combined selectable and scalable reporters that enable the sensitive screening of large peptide libraries followed by the sorting of positive interactions by the level of reporter output. We have applied this tool to characterize a set of human and E. coli PDZ domains. Our results are consistent with prior characterization of these proteins, and the improved sensitivity increases our ability to predict known and novel in vivo binding partners. This approach allows for the recovery of a wide range of affinities with a high throughput method that does not sacrifice the scale of the screen.
Assuntos
Escherichia coli/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Peptídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Genes Reporter , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Domínios PDZ , Biblioteca de Peptídeos , Peptídeos/química , Ligação ProteicaRESUMO
Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain-DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain-DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.
Assuntos
Cisteína/química , DNA/metabolismo , Histidina/química , Dedos de Zinco , Sítios de Ligação , DNA/química , Coleta de DadosRESUMO
Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.
Assuntos
Proteínas de Fluorescência Verde/genética , Engenharia de Proteínas/métodos , Biologia Sintética/métodos , Fatores de Transcrição/genética , Sequência de Bases , Conexina 43/genética , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Plasmídeos/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Leveduras/genética , Leveduras/metabolismoRESUMO
A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys(2)His(2) zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.