CRISPR Can Cut Proteins, Too

Cas7-11, in complex with a protein called Csx29, acts as an RNA-guided protease, meaning it can cleave other proteins.

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Never thought I’d write that headline. But a recent preprint shows that Cas7-11, in complex with a protein called Csx29, acts as an RNA-guided protease, meaning it can cleave other proteins. The results, which appeared last week on bioRxiv, will inevitably be adapted for programmable protein cleavage or to build new types of cell circuits.

No, not that kind of 7-11.

The Short: When Cas7-11 forms a complex with a protein called Csx29 (a protease), the two “cut” another protein, called Csx30, and split it into two. This protease activity is RNA-dependent.

Background: Cas7-11 is an RNA-guided enzyme that cuts single-stranded RNA. This enzyme consists of four Cas7 domains and a single Cas11 domain. When expressed in myriad organisms, it causes negligible cell toxicity.

  • The bacterium Desulfonema ishimotonii, first reported in 2000, has a genetic locus that encodes Cas7-11 and a slew of ‘accessory’ proteins, including Csx29, Csx30, and Csx31.
  • Cas7-11 was previously known to interact with Csx29, suggesting that it might be controlling the latter’s protease activity.

Experiments: The structure of Cas7-11 in complex with Csx29 was solved via cryo-EM, both with and without a 27-nucleotide crRNA and target RNA sequence, at 2.8 and 2.5-Å resolutions, respectively. CryoEM revealed that…

  • Cas7-11 is built from modular protein domains.
  • The Csx29 protein has a protease domain, called CHAT. Its protease activity is activated by Cas7-11.
  • When Cas7-11 forms a protein complex with Csx29 and a crRNA, the protease sits in its inactive state; its catalytic residue remains buried in the CHAT domain.

Rearrangements: When Cas7-11-crRNA-Csx29 pairs up with a target RNA sequence (say, genetic material from an invading virus), one of the Csx29 domains “dissociates from Cas7-11 and adopts a flexible conformation,” according to the preprint. This frees up the CHAT domain, which can then cut another protein.

  • Csx30 and Csx31 are encoded on the same genetic locus, all within a CRISPR operon.
  • The active form of Cas7-11-crRNA-Cxs29 cuts Csx30, in an RNA-dependent manner, between residues 427 and 428.
  • This cleavage produces toxic Csx30 fragments and leads to cell death. Thus, this RNA-directed protein cleavage serves as a bacterial defense system.

Why It Matters: Cas7-11 and Csx29 could perhaps be used for RNA-directed protein cleavage. This would have important applications in building biocontainment systems or to eliminate “cheater” strains during biomanufacturing.

Bio(tech) Index

(↑ = recommended article, * = open access, † = review, comment, etc. )

Basic Research

*Encapsulated bacteria deform lipid vesicles into flagellated swimmers. Le Nagard L…Staykova M. PNAS. Link

*Halogenation of tyrosine perturbs large-scale protein self-organization. Sun H…Budisa N. Nature Communications. Link

*A synthetic biology approach reveals diverse and dynamic CDK response profiles via multisite phosphorylation of NLS-NES modules. Faustova I…Loog M. Science Advances. Link

Increasing cell size remodels the proteome and promotes senescence. Lanz MC…Skotheim JM. Molecular Cell. Link

Biomanufacturing & Metabolic Engineering

↑Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide. Elashal HE…Link AJ. Nature Chemistry. Link

*Multi-sugar fermentation of lignocellulosic hydrolysate under industrially relevant conditions: comparison of several yeast strains. Froehlich A…Laine MM. bioRxiv (preprint). Link

Engineering microbial cell viability for enhancing chemical production by second codon engineering. Guo L…Chen X. Metabolic Engineering. Link

Evolution-Guided Biosynthesis of Terpenoid Inhibitors. Sarkar A…Fox JM. ACS Synthetic Biology. Link

Systematic Engineering of Escherichia coli for Efficient Production of Nicotinamide Mononucleotide From Nicotinamide. Huang Z…Zhou J. ACS Synthetic Biology. Link

Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid. Zhou Z…Yuan Q. Metabolic Engineering. Link

Thermodynamic limitations of PHB production from formate and fructose in Cupriavidus necator. Janasch M…Hudson EP. Metabolic Engineering. Link

Biosecurity

*†Biological Weapons Are a Thing of the Past…Or Are They? Lentzos F. GEN Biotechnology. Link

↑*†Evaluation of existing guidelines for their adequacy for the food and feed risk assessment of microorganisms obtained through synthetic biology. EFSA Scientific Committee. EFSA Journal. Link

Biosensors & Biomaterials

↑*Bioengineered textiles with peptide binders that capture SARS-CoV-2 viral particles. Navone L…Speight RE. Communications Materials. Link

  • Take some cotton. Build a protein that fuses a cellulose-binding domain to a peptide that binds the SARS-CoV-2 spike protein. Attach the two. What do you have? A “bioengineered cotton” that can capture over 100,000 virus particles per squared centimeter, and that “reduces onwards SARS-CoV-2 infection of cells by 500-fold.” A typical person with COVID-19 sheds about 120,000 viruses per milliliter of saliva, a concentration quite a bit lower than what can be absorbed by this material. The material could be embedded in face masks.

Computational Tools & Models

↑*†Deep Learning Concepts and Applications for Synthetic Biology. Beardall WAV, Stan G-B & Dunlop MJ. GEN Biotechnology. Link

*Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates. Watson ZL…Abramyan AM. bioRxiv (preprint). Link

*Defining hierarchical protein interaction networks from spectral analysis of bacterial proteomes. Zaydman MA…Raman AS. eLife. Link

*Experimental exploration of a ribozyme neutral network using evolutionary algorithm and deep learning. Rotrattanadumrong R & Yokobayashi Y. Nature Communications. Link

CRISPR & Gene Editing

*RNA-triggered protein cleavage and cell death by the RNA-guided type III-E CRISPR-Cas nuclease-protease complex. Kato K…Nishimasu H. bioRxiv (preprint). Link

↑*Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects. Kulcsár PI…Welker E. bioRxiv (preprint). Link

†What are the current bottlenecks in developing and applying CRISPR technologies? Kellogg EH…Qi LS. Cell Systems. Link

*Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803. Cengic I…Hudson EP. ACS Synthetic Biology. Link

Genetic Circuits

*Autoinhibited transient, gated, and cascaded dynamic transcription of RNAs. Li Z, Wang J & Willner I. Science Advances. Link

Medicine & Diagnostics

↑*Unbiased discovery of autoantibodies associated with severe COVID-19 via genome-scale self-assembled DNA-barcoded protein libraries. Credle JJ…Larman HB. Nature Biomedical Engineering. Link

*Treatment of allergic eosinophilic asthma through engineered IL-5-anchored chimeric antigen receptor T cells. Chen S…Ying S. Cell Discovery. Link

↑*Engineered Bacteriophages Containing Anti-CRISPR Suppress Infection of Antibiotic-Resistant P. aeruginosa. Qin S…Wu M. Microbiology Spectrum. Link

  • I’ve previously written about bacteriophages, and how they’ve been used to treat antibiotic-resistant lung infections. Now, bacteriophages are getting even better! In this paper, researchers packaged phages with anti-CRISPR genes (like AcrIF1, AcrIF2, and AcrIF3), and used them to destroy resistant Pseudomonas aeruginosa bacteria. The engineered phages were also injected into mice with clinically-isolated, multi-drug resistant P. aeruginosa bacteria, and these treatments increased survival rates, decreased bacterial counts, and reduced inflammation in the rodents.

*Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine. Arunachalam PS…Pulendran B. Science Translational Medicine. Link

*Adenovirus type 5 SARS-CoV-2 vaccines delivered orally or intranasally reduced disease severity and transmission in a hamster model. Langel SN…Tucker SN. Science Translational Medicine. Link

*Piperazine-derived lipid nanoparticles deliver mRNA to immune cells in vivo. Ni H…Dahlman JE. Nature Communications. Link

*Direct conversion of somatic cells into ‘insulin-producing-cells’ by user-defined multiplex-epigenetic-engineering vector (MEEV-β). Naqvi RA, Naqvi AR & Priyadarshini M. bioRxiv (preprint). Link

Plants

*Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system. Wang W…Akhunov E. Plant Biotechnology Journal. Link

Protein & Molecular Engineering

*Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Liu Z…St-Pierre F. Cell. Link

  • I’ve written a lot about two-photon calcium imagine. (I spent, like, 6 months working on this article). Genetically-encoded calcium sensors are a proxy for neural activity. But calcium fluctuations are really slow, and so people have long been working to make genetically-encoded voltage indicators (more details here), which sense the membrane potentials in a single cell and output fluorescence in response.

    An evolved protein, called JEDI-2P, is “faster, brighter. and more sensitive and photostable” than any other voltage indicator yet identified for two-photon microscopy. It can be used to record neural activity at depths exceeding 400 microns in a mouse brain, and can “optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min.” Amazing stuff.

*SNAP-tag and HaloTag fused proteins for HaSX8-inducible control over synthetic biological functions in engineered mammalian cells. Dotson HL & Ngo JT. bioRxiv (preprint). Link

*Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses. Schulz R…Siegert S. Nature Communications. Link

Generation of glycan-specific nanobodies. Khilji SK…Moscovitz O. Cell Chemical Biology. Link

*Extending Janus lectins architecture: characterization and application to protocells. Notova S…Imberty A. bioRxiv (preprint). Link

*Selecting better biocatalysts by complementing recoded bacteria. Rubini R…Mayer C. bioRxiv (preprint). Link

*De novo designed protein inhibitors of amyloid aggregation and seeding. Murray KA…Eisenberg DS. PNAS. Link

*Engineering an autonomous VH domain to modulate intracellular pathways and to interrogate the eIF4F complex. Frosi Y…Brown CJ. Nature Communications. Link

Tools & Technology

↑*Dynamic cybergenetic control of bacterial co-culture composition via optogenetic feedback. Gutiérrez Mena J, Kumar S & Khammash M. Nature Communications. Link

*Large scale screening of CRISPR guide RNAs using an optimized high throughput robotics system. Spangler JR…Stenger DA. Scientific Reports. Link

*Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies. Hartmann FSF…Seibold GM. bioRxiv (preprint). Link

*3D RNA-scaffolded wireframe origami. Parsons MF…Bathe M. bioRxiv (preprint). Link

*Live-seq enables temporal transcriptomic recording of single cells. Chen W…Deplancke B. Nature. Link

†Microfluidics: the propellant of CRISPR-based nucleic acid detection. Chen Y…Xu J. Trends in Biotechnology. Link

*DetecDiv, a generalist deep-learning platform for automated cell division tracking and survival analysis. Aspert T, Hentsch D & Charvin G. eLife. Link

Other…

*Proteome-scale induced proximity screens reveal highly potent protein degraders and stabilizers. Poirson J…Taipale M. bioRxiv (preprint). Link

*Precision Guided Sterile Males Suppress Populations of an Invasive Crop Pest. Kandul NP…Akbari OS. GEN Biotechnology. Link

*†Public Engagement with Biotechnology Inside and Outside the Classroom: Community-Focused Approaches. Jimenez J…Porras AM. GEN Biotechnology. Link

Rapid Nanopore Sequencing–Based Screen for Aneuploidy in Reproductive Care. The New England Journal of Medicine. Link


More soon,

— Niko McCarty

Twitter: @NikoMcCarty