6000+ Phages with CRISPR

More than 6,000 different viruses encode a CRISPR system. So when you hear about Cas9 and Cas12 and Cas13 and CasX, don’t stand with your mouth agape, amazed at humankind’s ingenuity in finding variants of the same thing.

Plus: A short history of beer.

This article is from my biotechnology newsletter, Codon. Subscribe for free.

An idyllic peace settles over my apartment. It’s the Sunday morning after Thanksgiving. My family is asleep, my cats lounge beneath sunbathed glass, and a gossamer thread of steam rises from my coffee mug. Life is good, writing is nice. Here’s to another year.

AI makes left-handed helices.

CRISPR is Ultra Common in Nature

More than 6,000 different viruses encode a CRISPR system. So when you hear about Cas9 and Cas12 and Cas13 and CasX, don’t stand with your mouth agape, amazed at humankind’s ingenuity in finding variants of the same thing. Instead, make a mental note: We barely understand anything about nature’s repertoire of DNA-editing tools.

These CRISPR proteins were discovered by using computers to pore over 660 billion base pairs of DNA collected from the environment and from the guts of animals. The CRISPR systems were found in bacteriophages, a type of virus that infects bacteria. Most of these phages have a genome size of around 52,000 base pairs, and each uses CRISPR in a slightly different way.

The researchers, from Jennifer Doudna’s group at UC Berkeley, also found the first examples of Cas13 proteins in phage. Cas13 proteins are unique because they can cut RNA, rather than DNA. Another family of Cas proteins, called Casλ, are also highly common in bacteriophages and are quite small, which means they can be packaged in viruses and delivered into mice or people with ease. When the Casλ phage proteins were directly compared to Cas12a in a head-to-head showdown (in HEK293T cells), the Casλ proteins “generated promising genome-editing outcomes compared to Cas12a, and in at least one case exceeded Cas12a insertion-deletion (indel) percentages.” Read more at Cell.

Mosquito Vaccines

A phase I trial with 51 participants found that a special type of vaccine triggered immune responses against malarial pathogens in healthy adults, aged 18 to 50 years old.

A “mosquito salivary peptide,” called AGS-v PLUS, the vaccine is designed to protect against multiple mosquito-borne diseases at once. Participants were split into five different groups. Some people received two doses of placebo, while others got two doses of the vaccine or one dose of each; placebo and vaccine.

The vaccine is a concoction of five synthetic peptides found in the saliva of different mosquito species. Each participant was injected with placebo or the vaccines on days 1 and 22. Various metabolites, like IgG, IgM and IgE antibodies, as well as interferon-ɣ and interleukin-4, were measured in each study participant on days 1, 43, and 50. None of the participants had a serious, adverse event.

On day 43, each participant was feasted upon my mosquitoes. Specifically, Aedes aegypti — which can spread dengue, chikungunya and Zika fever — and Aedes albopictus — which can spread chikungunya virus, dengue virus and dirofilariasis —were placed in containers and then set upon each participant’s arms for 10 to 20 minutes. Skin redness was measured 30 minutes later to see whether those with the vaccine had less redness. The “bite site swelling diameter was not statistically different for any group,” but those who received the vaccine did have significantly increased IFN-ɣ levels in response to AGS-v PLUS antigens, suggesting that the vaccine triggered an immune response and could (possibly) help combat Zika virus and other mosquito-borne diseases. A phase II trial is probably in the works. Read more at eBioMedicine.

Beer’s Past

Every once in a while, I sit down with a glass of wine (or beer) and read through a research review. It sounds super lame, but some are actually well-written and teach me things about adjacent research topics, which means they’re actually fun to read.

So here’s some fun facts that I picked up from reading this new review about beer brewing’s history.

  1. The earliest beers, crafted at least 6,000 years ago, were probably created by “chewing grain and salivating” to turn cereals into sugar. This is still used to make chicha, a fermented corn beverage common in Peru.
  2. Saccharomyces cerevisiae, aka Baker’s Yeast, was domesticated much like cows and dogs. Humans have made it the most successful yeast organism on planet Earth because, somehow, it evolved the ability to metabolize “malt sugar, which is one of the rarest sugars in the wild.” WTF?
  3. Ancient peoples added bark, honey, and fruit juices to flavor their beers, much as we do today.
  4. “Brewing beer in a warm climate is more difficult because fermentation proceeds faster and more uncontrolled, the risk of infection by beer spoilage organisms increases and shelf-life decreases. It was therefore an extraordinary achievement by the first brewers in the Fertile Crescent and ancient Egypt to develop and improve the fermentation process to mass-produce beer with a consistent quality.” First the pyramids and now this. Aliens?
  5. Until the Middle Ages, monasteries played a major role in passing down brewing knowledge from one generation to the next. Monks kept “meticulous records” of brewing experiments. And yeast were not identified as the “fermenting organism” until the middle of the 19th century, nearly 200 years after Antoni van Leeuwenhoek first discovered microbes! Read more at EMBO Reports.

Thanks for reading.

— Niko

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The Index 🔻

A collection of research papers published in the last week.

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

Basic Research

Ancient origin and constrained evolution of the division and cell wall gene cluster in Bacteria. Megrian D…Gribaldo S. Nature Microbiology. Link

*Resource sharing is sufficient for the emergence of division of labour. Kreider JJ…Weissing FJ. Nature Communications. Link

Antiviral signaling by a cyclic nucleotide activated CRISPR protease. Rouillon C…Hagelueken G. Nature. Link

*Probing the genetic code and impacts of mistranslation using tRNA-Ala anticodon variants. Cozma E…Berg MD. bioRxiv (preprint). Link

*Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system. Shiriaeva AA…Severinov K. Science Advances. Link

*Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis. Yan M-Y…Sun Y-C. Science Advances. Link


*Engineering stringent genetic biocontainment of yeast with a protein stability switch. Hoffmann SA & Cai Y. bioRxiv (preprint). Link

*Synthetic engineering and biological containment of bacteriophages. Mitsunaka S…Ando H. PNAS. Link

Biomanufacturing, Metabolic Engineering & Biomaterials

↑*Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides. Pandi A…Erb TJ. bioRxiv (preprint). Link

*Enhanced production of acetyl-CoA-based products via peroxisomal surface display in Saccharomyces cerevisiae. Yocum HC, Bassett S & Da Silva NA. PNAS. Link

*Development of Expression-Tunable Multiple Protein Syntheses System in Cell-Free Reaction using T7-promoter-variant Series. Senda N…Nishida H. Synthetic Biology. Link

*Biotechnologically produced chitosans with nonrandom acetylation patterns differ from conventional chitosans in properties and activities. Streekumar S…Moerschbacher BM. Nature Communications. Link

Base editing for reprogramming cyanobacterium Synechococcus elongatus. Wang S-Y…Xia P-F. Metabolic Engineering. Link

Poly-γ-Glutamic Acid Production by Engineering a DegU Quorum-Sensing Circuit in Bacillus subtilis. Hu L-X…Liu Y. ACS Synthetic Biology. Link

Cell-Free Protein Synthesis of Particulate Methane Monooxygenase into Nanodiscs. Koo CW…Rosenzweig AC. ACS Synthetic Biology. Link

*A termination-aware vector design improves heterologous gene expression in Pseudomonas putida. de Siqueira GMV & Guazzaroni M-E. bioRxiv (preprint). Link

Computational Tools & Models

↑*AlphaFill: enriching AlphaFold models with ligands and cofactors. Hekkelman ML…Perrakis A. Nature Methods. Link

  • I didn’t even know (though I guess it’s reasonable to assume) that all the millions of AlphaFold models released by DeepMind don’t have any small molecules associated with them. The AI-generated structures are just 3-D models of proteins floating alone, in isolation. A new paper has rectified this. AlphaFill is “an algorithm that uses sequence and structure similarity to ‘transplant’ such ‘missing’ small molecules and ions from experimentally determined structures to predicted protein models.” Seems like it could be somewhat useful for, say, predicting the functions of enzymes in strange microbes, or piecing together unknown metabolic pathways.

*proGenomes3: approaching one million accurately and consistently annotated high-quality prokaryotic genomes. Fullam A…Mende DR. Nucleic Acids Research. Link

*Deep reinforcement learning for optimal experimental design in biology. Treloar NJ…Barnes CP. PLoS Computational Biology. Link

*Sampling of structure and sequence space of small protein folds. Linsky TW…Strauch EM. Nature Communications. Link

Prediction of the cell-type-specific transcription of non-coding RNAs from genome sequences via machine learning. Koido M…Terao C. Nature Biomedical Engineering. Link

*AlphaPeptDeep: a modular deep learning framework to predict peptide properties for proteomics. Zeng W-F…Mann M. Nature Communications. Link

*DrugnomeAI is an ensemble machine-learning framework for predicting druggability of candidate drug targets. Raies A…Vitsios D. Communications Biology. Link

*SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes. Martínez-García E…de Lorenzo V. Nucleic Acids Research. Link

*GPSAdb: a comprehensive web resource for interactive exploration of genetic perturbation RNA-seq datasets. Guo S…Song W. Nucleic Acids Research. Link

CRISPR, DNA Editing & Regulation

↑*Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors. Al-Shayeb B…Doudna JA. Cell. Link

↑*Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo. Willis JCW…Liu DR. Nature Communications. Link

  • It’s really hard to engineer mitochondrial DNA with CRISPR (the guide RNA can’t get into the organelle), but zinc finger base editors work just fine. A new study made tiny enzymes that have “improved DNA specificity” and can “install or correct disease-associated mutations in mitochondria.” They worked well in mice, and made targeted edits in the heart, liver, and skeletal muscle.

↑Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Yarnall MTN…Gootenberg JS. Nature Biotechnology. Link

The miniature CRISPR-Cas12m effector binds DNA to block transcription. Wu WY…van der Oost J. Molecular Cell. Link

Three novel Cas12a orthologs with robust DNA cleavage activity suitable for nucleic acid detection. Liu X…Li Z. Gene. Link

*A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias. Verkuijl SAN…Alphey L. Nature Communications. Link

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi. Rostain W…Tolonen AC. ACS Synthetic Biology. Link

Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei. Gu S…Zhong J. ACS Synthetic Biology. Link

*Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria. Dolcemascolo R…Rodrigo G. bioRxiv (preprint). Link

*CRISPRi in Deinococcus radiodurans. Misra CS…Rath D. bioRxiv (preprint). Link

DNA Sequencing, Synthesis & Assembly

m6A-SAC-seq for quantitative whole transcriptome m6A profiling. Ge R…He C. Nature Protocols. Link

*Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging. Haase C…Lin CP. Nature Methods. Link

*A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation. Strawn IK…Whitehead TA. bioRxiv (preprint). Link

*Enzymatic assembly of small synthetic genes with repetitive elements. Nguyen MTA…Andersen ES. bioRxiv (preprint). Link

Genetic Circuits

↑*Periodic spatial patterning with a single morphogen. Wang S, Garcia-Ojalvo J & Elowitz MB. Cell Systems. Link

  • This one may change your outlook on the “special” status ascribed to life. You can make spatially periodic patterns during multicellular development using “only a single diffusible morphogen,” according to a computational model. This is an enticing starting point for newfangled experiments in “synthetic developmental biology.” To understand how an embryo becomes a baby, let’s recreate the process from scratch.

†Toward predictive engineering of gene circuits. Şimşek E…You L. Trends in Biotechnology. Link

*Developing a temperature-inducible transcriptional rheostat in Neurospora crassa. Tabilo-Agurto C…Larrondo LF. bioRxiv (preprint). Link

Medicine & Diagnostics

↑Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy. Nishiyama T…Olson EN. Science Translational Medicine. Link

  • This is a big one. A base editor corrected a mutation (R634Q) underlying an inherited heart disease — dilated cardiomyopathy. It worked in cell culture (with about 40% efficiency) and in mice carrying this mutation. For the latter, it was delivered via AAV9 and extended life spans.

↑*Safety and immunogenicity of AGS-v PLUS, a mosquito saliva peptide vaccine against arboviral diseases: A randomized, double-blind, placebo-controlled Phase 1 trial. Friedman-Klabanoff DJ…Laurens MB. eBioMedicine. Link

↑*An automated DNA computing platform for rapid etiological diagnostics. Ma Q…Tan W. Science Advances. Link

↑*Immune engineered extracellular vesicles to modulate T cell activation in the context of type 1 diabetes. Becker MW…Phelps EA. bioRxiv (preprint). Link

*Unadjuvanted intranasal spike vaccine elicits protective mucosal immunity against sarbecoviruses. Mao T…Iwasaki A. Science. Link

*A potent and broad neutralization of SARS-CoV-2 variants of concern by DARPins. Chonira V…Chen Z. Nature Chemical Biology. Link

An ultrasensitive and specific ratiometric electrochemical biosensor based on SRCA-CRISPR/Cas12a system for detection of Salmonella in food. Zheng S…Zhang W. Food Control. Link


↑Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs). Jin S…Gao C. Nature Protocols. Link

  • It’s not often that I see new papers about plant gene editing. Maybe I’m looking in the wrong places. A new protocol outlines a tool, called PlantPegDesigner, that can quickly design prime editing “vectors with enhanced editing efficiency.” The predictive model is based on data collected from rice. The full process to make the prime editing vector, design the guide RNAs, and get everything into a plant takes between 4 and 7 days.

*A novel plant-made monoclonal antibody enhances the synergetic potency of an antibody cocktail against the SARS-CoV-2 Omicron variant. Jugler C…Chen Q. Plant Biotechnology Journal. Link

Tobacco rattle virus-induced VcANS gene silencing in blueberry fruit. Tian L…Yu H. Gene. Link

*CRISPR-induced miRNA156-recognition element mutations in TaSPL13 improve multiple agronomic traits in wheat. Gupta A…Li W. Plant Biotechnology Journal. Link

†Trichoderma: a multipurpose, plant-beneficial microorganism for eco-sustainable agriculture. Woo SL…Monte E. Nature Reviews Microbiology. Link

†Genome Editing in Plants. Wada N, Osakabe K & Osakabe Y. Gene and Genome Editing. Link

Protein & Molecular Engineering

↑*Accurate Computational Design of 3D Protein Crystals. Li Z…Baker D. bioRxiv (preprint). Link

Proteins that self-assemble into large, three-dimensional crystals were designed on a computer, fabricated in the lab, and then actually folded as expected in the real world. Changing amino acids at the atomic level can give rise to crystals, larger than 100 micrometers in size, with exquisitely distinct geometries.

From Li Z et al. “Accurate Computational Design of 3D Protein Crystals” in bioRxiv

*High-performance optical control of GPCR signaling by bistable animal opsins MosOpn3 and LamPP in a molecular property–dependent manner. Koyanagi M…Terakita A. PNAS. Link

*Structure- and computational-aided engineering of an oxidase to produce isoeugenol from a lignin-derived compound. Guo Y…Fraaije MW. Nature Communications. Link

*A general approach for stabilizing nanobodies for intracellular expression. Dingus JG…Cepko CL. eLife. Link

†Engineering Rieske oxygenase activity one piece at a time. Brimberry M…Bridwell-Rabb J. Current Opinion in Chemical Biology. Link

*Synthetic nanobodies as tools to distinguish IgG Fc glycoforms. Kao KS…Ravetch JV. PNAS. Link

Tissue Engineering

Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes. Xu J…Shao Z. Nature Biomedical Engineering. Link

Tools, Toolkits & Technology

High-throughput retrieval of target sequences from complex clone libraries using CRISPRi. Burian J…Brady SF. Nature Biotechnology. Link

*Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine. Moen JM…Rinehart J. Nature Communications. Link

*Controlling the composition of bacterial co-cultures via in silico feedback. Thesis from ETZ Zurich, by Joaquín Gutiérrez. Link

*TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages. Espinosa-Medina I…Lee T. Neuron. Link

Other Interesting Stuff

↑*A short history of beer brewing. Raihofer L…Hutzler M. EMBO Reports. Link

*Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. GBD 2019 Antimicrobial Resistance Collaborators. The Lancet. Link

  • Staphylococcus aureus and E. coli bacteria kill more people than any other bacterial pathogen — about 1 million people per year each. Infections in the thorax, and those in the bloodstream, are the leading causes. If you want to make a difference, look no further than this.

*Interpreting and de-noising genetically engineered barcodes in a DNA virus. Blois S…Sullivan CS. PLoS Computational Biology. Link

Metal-binding proteins and cross-linking in the defensive glue of the slug Arion subfuscus. Christoforo C…Smith AM. Journal of the Royal Society Interface. Link

Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching. Kosmidis E…Stamou D. Nature. Link

*Ancient oral microbiomes support gradual Neolithic dietary shifts towards agriculture. Quagliariello A…Lari M. Nature Communications. Link