CRISPR madness
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Rescooped by Anna Nikulina from Protein folds and folding
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Expanding Anfinsen’s Principle: Contributions of Synonymous Codon Selection to Rational Protein Design

Expanding Anfinsen’s Principle: Contributions of Synonymous Codon Selection to Rational Protein Design | CRISPR madness | Scoop.it

Abstract

Anfinsen’s principle asserts that all information required to specify the structure of a protein is encoded in its amino acid sequence. However, during protein synthesis by the ribosome, the N-terminus of the nascent chain can begin to fold before the C-terminus is available. We tested whether this cotranslational folding can alter the folded structure of an encoded protein in vivo, versus the structure formed when refolded in vitro. We designed a fluorescent protein consisting of three half-domains, where the N- and C-terminal half-domains compete with each other to interact with the central half-domain. The outcome of this competition determines the fluorescence properties of the resulting folded structure. Upon refolding after chemical denaturation, this protein produced equimolar amounts of the N- and C-terminal folded structures, respectively. In contrast, translation in Escherichia coli resulted in a 2-fold enhancement in the formation of the N-terminal folded structure. Rare synonymous codon substitutions at the 5′ end of the C-terminal half-domain further increased selection for the N-terminal folded structure. These results demonstrate that the rate at which a nascent protein emerges from the ribosome can specify the folded structure of a protein.


Via Bernard Offmann
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Bernard Offmann's curator insight, February 23, 2014 9:48 AM

This very nice paper was quoted by Catherine Goodman in Nature Chemical Biology (Protein Folding; The Inside Scoop).(http://www.nature.com/nchembio/journal/v10/n3/full/nchembio.1465.html)

It shows elegantly how codon usage can affect folding.

Rescooped by Anna Nikulina from Biomedical synthetic biology
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Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing | CRISPR madness | Scoop.it

The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.


Via omni
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omni's curator insight, October 5, 2013 2:38 AM

George Church's paper.

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Improving CRISPR-Cas nuclease specificity using truncated guide RNAs : Nature Biotechnology : Nature Publishing Group

Improving CRISPR-Cas nuclease specificity using truncated guide RNAs : Nature Biotechnology : Nature Publishing Group | CRISPR madness | Scoop.it
Guide RNAs with shorter regions of complementarity to target sites reduce off-target effects.
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Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system | CRISPR madness | Scoop.it
Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.
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Rescooped by Anna Nikulina from Amazing Science
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Crystallographic elucidation of CRISPR-Cas9 endonucleases reveal RNA-mediated conformational changes

Crystallographic elucidation of CRISPR-Cas9 endonucleases reveal RNA-mediated conformational changes | CRISPR madness | Scoop.it

The potential is there for bacteria and other microbes to be genetically engineered to perform a cornucopia of valuable goods and services, from the production of safer, more effective medicines and clean, green, sustainable fuels, to the clean-up and restoration of our air, water and land. Cells from eukaryotic organisms can also be modified for research or to fight disease. To achieve these and other worthy goals, the ability to precisely edit the instructions contained within a target's genome is a must. A powerful new tool for genome editing and gene regulation has emerged in the form of a family of enzymes known as Cas9, which plays a critical role in the bacterial immune system. Cas9 should become an even more valuable tool with the creation of the first detailed picture of its three-dimensional shape by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley.

 

Biochemist Jennifer Doudna and biophysicist Eva Nogales, both of whom hold appointments with Berkeley Lab, UC Berkeley, and the Howard Hughes Medical Institute (HHMI), led an international collaboration that used x-ray crystallography to produce 2.6 and 2.2 angstrom (Å) resolution crystal structure images of two major types of Cas9 enzymes. The collaboration then used single-particle electron microscopy to reveal how Cas9 partners with its guide RNA to interact with target DNA. The results point the way to the rational design of new and improved versions of Cas9 enzymes for basic research and genetic engineering.

Cas9 is a family of RNA-guided bacterial endonucleases employed by Type II CRISPR systems to recognize and cleave double-stranded DNA at site-specific sequences. Genetic engineers have begun harnessing Cas9 for genome editing and gene regulation in many eukaryotic organisms. However, despite the successes to date, the technology has yet to reach its full potential because until now the structural basis for guide RNA recognition and DNA targeting by Cas9 has been unknown.

 

What has been a major puzzle in the CRISPR–Cas field is how Cas9 and similar RNA-guided complexes locate and recognize matching DNA targets in the context of an entire genome, the classic needle in a haystack problem," says Samuel Sternberg, lead author of the Nature paper and a member of Doudna's research group. "All of the scientists who are developing RNA-programmable Cas9 for genome engineering are relying on its ability to target unique 20-base-pair long sequences inside the cell. However, if Cas9 were to just blindly bind DNA at random sites across a genome until colliding with its target, the process would be incredibly time-consuming and probably too inefficient to be effective for bacterial immunity, or as a tool for genome engineers. Our study shows that Cas9 confines its search by first looking for PAM sequences. This accelerates the rate at which the target can be located, and minimizes the time spent interrogating non-target DNA sites."

 

Now, several scientists addressed this lack of detailed knowledge about Cas9 by first solving the three-dimensional crystal structures of two Cas9 proteins, representing large and small versions, from Streptococcus pyogenes (SpyCas) and Actinomyces naeslundii (AnaCas9) respectively. Using protein crystallography beamlines at Berkeley Lab's Advanced Light Source and the Paul Scherer Institute's Swiss Light Source, the collaboration discovered that despite significant differences outside of their catalytic domains, all members of the Cas9 family share the same structural core. The high resolution images showed this core to feature a clam-shaped architecture with two major lobes - a nuclease domain lobe and an alpha-helical lobe. Both lobes contained conserved clefts that become functional in nucleic acid binding.

 

"Our understanding of Cas9's structure was not complete with only the x-ray data because the protein in the crystals had been trapped in a state without its associated guide RNA," says Sam Sternberg, a member of Doudna's research group and a co-author of the Science paper. "Understanding how RNA-guided Cas9 targets matching DNA sequences for genome engineering and how this reaction and its specificity might be improved required an understanding of how the shape of Cas9 changes when it interacts with guide RNA, and when a matching DNA target sequence is bound."

 

The collaboration employed negative-staining electron microscopy to visualize the Cas9 protein bound to either guide RNA, or both RNA and target DNA. The structures revealed that the guide RNA binding structurally activates Cas9 by creating a channel between the two main lobes of the protein that functions as the DNA-binding interface.

 

"Our single particle electron microscopy analysis reveals the importance of guide-RNA for the conversion of Cas9 into a structurally-activated state," says David Taylor, a joint member of Doudna's and Nogales's research groups and another co-author of the Science paper. "The results underline that, in addition to sequence complementarity, other features of the guide-RNA must be considered when employing this technology."


Via Dr. Stefan Gruenwald
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Shortening guide RNA markedly improves specificity of CRISPR-Cas nucleases - Biology News Net (press release)

Genetic Engineering News Shortening guide RNA markedly improves specificity of CRISPR-Cas nucleases Biology News Net (press release) CRISPR-Cas RGNs combine a gene-cutting enzyme called Cas9 with a short RNA segment and are used to induce breaks in...
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