A New Family of CRISPR Proteins

Cas12l prefers to cut supercoiled DNA.

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Newly unearthed CRISPR proteins, discovered in a little-known phylum of gram-negative bacteria called Armatimonadetes, have been dubbed Cas12l. The reason is simple: Scientists have already discovered Cas12a-k.

But these new proteins are worth noting for a few reasons. Much like other Cas proteins, they are guided to a DNA target by a short piece of RNA, measuring 20 nucleotides in length. But unlike similar proteins, which cut DNA near T-rich PAM sequences — the short motif near a DNA target that is required for recognition — Cas12l cleaves near C-rich PAM sequences (specifically CCY, where Y is either C or T).

These proteins are also quite small, making it more likely they’ll fit inside of viruses that are commonly used for gene therapies. Tested Cas12l proteins measure 860 amino acids in length. Cas12a (the most commonly used Cas12 protein) is big; between 1200 and 1500 amino acids.

Panel A shows the genetic loci for various Cas12l proteins. The accessory proteins Cas1, Cas2 and Cas4 are all present. Panel B shows the broad diversity of Cas12 proteins, which now extends through family ‘L’!

After cleaving its target DNA, Cas12l also goes on a brief hunting spree, indiscriminately chopping up nearby single-stranded DNA and RNA sequences. This feature could be exploited for future diagnostics, much like Cas12a has been.

A specific version of Cas12l, called Asp2Cas12l, was also expressed in E. coli bacteria and, once inside, successfully chopped up double-stranded DNA. This suggests that Cas12l is portable — it can be used in a broad lineup of organisms for genetic engineering.

The new study was spearheaded by Tomas Urbaitis and Virginijus Šikšnys at Vilnius University in Lithuania. Šikšnys was among the first scientists to demonstrate that Cas9 can be programmed to cut DNA. He submitted an article on that subject to Cell Reports on April 18th, 2012. It was desk rejected by the editor. Šikšnys re-submitted the paper to PNAS one month later, on May 21st, and it took several months for review and publication.

On June 8th of that year, Martin Jinek, Jennifer Doudna, and Emmanuelle Charpentier submitted their famous paper, “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity,” to Science. It was (rightfully) accepted just 12 days later.

Thanks for reading.

— Niko McCarty

A silkworm, called Bombyx mori, makes the majority of our planet’s silk. About 146,000 tons are made in China alone each year. This insect’s silk is strong, tough, and light. A single bug can synthesize 20-35% of its own weight in silk in a single week. For a new study, researchers made transgenic silkworms — that is, they inserted new genes into the bugs — that cause them to excrete ‘supercharged’ silk proteins. The engineered silk was stronger (more tolerant of stress), had a higher crystallinity, and released moisture faster than normal silk. Nature Communications (Link)

A gene called C9ORF72 encodes a protein that is important for transporting other proteins within a cell. Sometimes a specific string of DNA — GGGGCC — is inserted many times within this gene. Such insertions are the most common genetic cause of amyotrophic lateral sclerosis, or ALS, a devastating and incurable condition that causes nerve cells to slowly break down over time. For a new study, the gene-editing tool, CRISPR-Cas9, was used to remove the repetitive DNA from C9ORF72, both in neurons grown in cell culture and in the brains of living mice. The genetic excision reduced some cellular hallmarks that are commonly associated with ALS, but much more research is needed before this therapeutic tool is tested in people or even chimps. Nature Communications (Link)

Men produce about 10% more 1st author papers — and collect 17% more citations — compared to their female counterparts. The discrepancy — based on an analysis of 42,000 PhD students at 235 institutions in the U.S. — persists even after matching graduate students by program and advisor. This effect disappeared when women joined labs with a same-gender advisor — and about 58% did exactly that. bioRxiv (Link)

Table 2 from “The Gender Gap Amongst Doctoral Students in the Biomedical” by Michael D. Schaller.

The Index 🔻

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

Basic Research

*Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform. Siepe DH…Garcia KC. eLife. Link

*Proteome effects of genome-wide single gene perturbations. Öztürk M…Butter F. Nature Communications. Link

Base editor scanning charts the DNMT3A activity landscape. Lue NZ…Liau BB. Nature Chemical Biology. Link

*Contribution of CRISPRable DNA to human complex traits. Zhai R…Shen X. Communications Biology. Link

Biomanufacturing, Metabolic Engineering & Biomaterials

*Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm. Chen X…Xu S. Nature Communications. Link

↑*Engineered synthetic one-carbon fixation exceeds yield of the Calvin Cycle. Dronsella B…Claassens NJ. bioRxiv (preprint). Link

*Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk. Fan R…Aranko AS. bioRxiv (preprint). Link

*Microbial nanowires with genetically modified peptide ligands to sustainably fabricate electronic sensing devices. Lekbach Y…Lovley DR. bioRxiv (preprint). Link

*Design of programmable post-translational switch control platform for on-demand protein secretion in mammalian cells. Mansouri M…Fussenegger M. Nucleic Acids Research. Link

*Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO2 Fixation. Davison PA…Huang WE. ACS Synthetic Biology. Link

*†Harnessing bioengineered microbes as a versatile platform for space nutrition. Llorente B…Paulsen IT. Nature Communications. Link

Systems engineering of Escherichia coli for n-butane production. Liu Y…Mahadevan R. Metabolic Engineering. Link

*Geometrically programmed self-limited assembly of tubules using DNA origami colloids. Hayakawa D…Rogers WB. PNAS. Link

*Chemical fuel-driven dissipative living materials. Jo H…Sim S. ChemRxiv (preprint). Link

†Bioeconomy and net-zero carbon: lessons from Trends in Biotechnology, volume 1, issue 1. Philp J. Trends in Biotechnology. Link

*Engineering ssRNA tile filaments for (dis)assembly and membrane binding. De Franceschi N, Hoogenberg B & Dekker C. bioRxiv (preprint). Link


↑*Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts. Jensen ED…Jensen MK. Nature Communications. Link

*Generalized strategy for engineering mammalian cell-compatible RNA-based biosensors from random sequence libraries. Allchin ER…Lippmann ES. bioRxiv (preprint). Link

A propeptide-based biosensor for the selective detection of Vibrio cholerae using an environment-sensitive fluorophore. DeColli AA…Hatzios SK. Cell Chemical Biology. Link

*Enhanced small green fluorescent proteins as a multisensing platform for biosensor development. Liang G-T…Piatkevich KD. Frontiers in Bioengineering and Biotechnology. Link

*High performance genetically-encoded green fluorescent biosensors for intracellular L-lactate. Le GNT…Campbell RE. bioRxiv (preprint). Link


↑Refactored genetic codes enable bidirectional genetic isolation. Zürcher JF…Chin JW. Science. Link

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles. Chauhan G…Abel SM. ACS Synthetic Biology. Link

Computational Tools & Models

*A generic framework for hierarchical de novo protein design. Harteveld Z…Correia BE. PNAS. Link

*Improved AlphaFold modeling with implicit experimental information. Terwilliger TC…Adams PD. Nature Methods. Link

*Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain. Nguyen PT…Yarov-Yarovoy V. eLife. Link

*Combining mass spectrometry and machine learning to discover bioactive peptides. Madsen CT…de Lichtenberg U. Nature Communications. Link

*Rosetta’s Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery. Okwei E…Meiler J. bioRxiv (preprint). Link

Resistor: An algorithm for predicting resistance mutations via Pareto optimization over multistate protein design and mutational signatures. Guerin N…Donald BR. Cell Systems. Link

PyImageJ: A library for integrating ImageJ and Python. Rueden CT…Eliceiri KW. Nature Methods. Link

*Aquila: a spatial omics database and analysis platform. Zheng Y…Cheung E. Nucleic Acids Research. Link

CRISPR, Gene Editing & Control

↑*A new family of CRISPR-type V nucleases with C-rich PAM recognition. Urbaitis T…Siksnys V. EMBO Reports. Link

↑*A synthetic transcription platform for programmable gene expression in mammalian cells. Chen WCW…Lu TK. Nature Communications. Link

*Comparison of DNA targeting CRISPR editors in human cells. Huang H…Lin Y. bioRxiv (preprint). Link

*ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time. Malcı K…Rios-Solis L. ACS Synthetic Biology. Link

DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9. Shi Y-J…Zhang Y. Molecular Cell. Link

*CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery. Benns HJ…Child MA. Nature Microbiology. Link

*G-quadruplex-guided RNA engineering to modulate CRISPR-based genomic regulation. Liu X…Zhou X. Nucleic Acids Research. Link

*Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing. Park SH…Bao G. Science Advances. Link

DNA Sequencing, Synthesis & Assembly

*SLICER: Seamless Loss of Integrated Cassettes Using Endonuclease Cleavage and Recombination in Deinococcus radiodurans. Brumwell SL…Karas BJ. bioRxiv (preprint). Link

Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins. Szent-Gyorgyi C…van de Weerd R. ACS Synthetic Biology. Link

Gene Circuits

↑*Resource-aware construct design in mammalian cells. Roberto DB…Francesca C. bioRxiv (preprint). Link

*Toehold-Mediated Strand Displacement in Random Sequence Pools. Mayer T, Oesinghaus L & Simmel FC. bioRxiv (preprint). Link

Medicine & Diagnostics

↑*CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. Meijboom KE…Mueller C. Nature Communications. Link

↑*Synthetic biology-instructed transdermal microneedle patch for traceable photodynamic therapy. He G…Huang P. Nature Communications. Link

*Bipolar cell targeted optogenetic gene therapy restores parallel retinal signaling and high-level vision in the degenerated retina. Kralik J…Kleinlogel S. Communications Biology. Link

*ChAd155-RSV vaccine is immunogenic and efficacious against bovine RSV infection-induced disease in young calves. de Jong R…Steff A-M. Nature Communications. Link

*Suppression of heterotopic ossification in fibrodysplasia ossificans progressiva using AAV gene delivery. Yang Y-S…Shim J-H. Nature Communications. Link

†Targeting inflammation to improve regulatory T cell therapy for immunopathologies. Salomon BL. PNAS. Link

*CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo. Wu J…Hu J. Nucleic Acids Research. Link

*PAM-less Exonuclease-assisted Cas12a for visual detection of Vibrio Species. Zhang DH, Raykar S & Ng KTC. bioRxiv (preprint). Link

*Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum. Iwanaga S…Uthaipibull C. Nature Communications. Link

*Adaptation to host cell environment during experimental evolution of Zika virus. Grass V…Dreux M. Communications Biology. Link

Genome-wide CRISPR screens identify combinations of candidate latency reversing agents for targeting the latent HIV-1 reservoir. Dai W…Siliciano RF. Science Translational Medicine. Link


*In vivo visualization of nitrate dynamics using a genetically encoded fluorescent biosensor. Chen Y-N, Cartwright HN & Ho C-H. Science Advances. Link

Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana. Cody JP…Voytas DF. Nature Protocols. Link

†CRISPR-based point-of-care plant disease diagnostics. Islam T & Kasfy SH. Trends in Biotechnology. Link

*PlantExp: a platform for exploration of gene expression and alternative splicing based on public plant RNA-seq samples. Liu J…Dou D. Nucleic Acids Research. Link

*†Linking plant functional genes to rhizosphere microbes: a review. Liu Q…Lian T. Plant Biotechnology Journal. Link

Tissue, Protein & Molecular Engineering

*Nature-inspired engineering of an artificial ligase enzyme by domain fusion. Tong CL…Seelig B. Nucleic Acids Research. Link

*Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast. Lahiri P & Van Deventer JA. bioRxiv (preprint). Link

†The molecular basis and enzyme engineering strategies for improvement of coupling efficiency in cytochrome P450s. Meng S…Schwaneberg U. Biotechnology Advances. Link

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail. Tristan CA…Singeç I. Nature Protocols. Link

*Directed evolution of aerotolerance in sulfide-dependent thiazole synthases. Van Gelder K…Hanson AD. bioRxiv (preprint). Link

*Ex vivo engineered human plasma cells exhibit robust protein secretion and long-term engraftment in vivo. Cheng R.Y-H…James RG. Nature Communications. Link

Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds. Meran L…Li VSW. Nature Protocols. Link

Tools & Technology

↑*A scalable platform to discover antimicrobials of ribosomal origin. Ayikpoe RS…Zhao H. Nature Communications. Link

*Spatially resolved epigenomic profiling of single cells in complex tissues. Lu T, Ang CE & Zhuang X. Cell. Link

*A Vaccinia-based system for directed evolution of GPCRs in mammalian cells. Klenk C…Plückthun A. bioRxiv (preprint). Link

Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID. Kwak C…Rhee H-W. Cell Chemical Biology. Link

Optimized single-nucleus transcriptional profiling by combinatorial indexing. Martin BK…Shendure J. Nature Protocols. Link

EasyGuide Plasmids Support in Vivo Assembly of gRNAs for CRISPR/Cas9 Applications in Saccharomyces cerevisiae. Jacobus AP…Gross J. ACS Synthetic Biology. Link

*Evaluation of a library of loxP variants with a wide range of recombination efficiencies by Cre. Yamauchi Y…Aoki W. PLOS ONE. Link

A microfluidic Braille valve platform for on-demand production, combinatorial screening and sorting of chemically distinct droplets. Utharala R…Merten CA. Nature Protocols. Link

Other Interesting Stuff

↑Nonlinear decision-making with enzymatic neural networks. Okumura S…Genot AJ. Nature. Link

↑*The Gender Gap Amongst Doctoral Students in the Biomedical Sciences. Schaller MD. bioRxiv (preprint). Link

*Information decay and enzymatic information recovery for DNA data storage. Meiser LC…Grass RN. Communications Biology. Link

*†The patent dispute over the breakthrough mRNA technology. Aquino-Jarquin G. Frontiers in Bioengineering and Biotechnology. Link

*Borgs are giant genetic elements with potential to expand metabolic capacity. Al-Shayeb B…Banfield JF. Nature. Link

*Hurdles in responsive community engagement for the development of environmental biotechnologies. Normandin AM…Evans SW. Synthetic Biology. Link

*Evolution of immune genes is associated with the Black Death. Klunk J…Barreiro LB. Nature. Link

*Long-term whole blood DNA preservation by cost-efficient cryosilicification. Zhou L…Zhu W. Nature Communications. Link

*Severe testing with high-dimensional omics data for enhancing biomedical scientific discovery. Emmert-Streib F. npj Systems Biology and Applications. Link