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| Open AccessLatent generative landscapes as maps of functional diversity in protein sequence space
In this work, the authors study protein families’ VAE latent manifolds and coevolutionary Hamiltonians. These Latent Generative Landscapes predict phylogenetic groupings, fitness & functional properties for several systems with clear protein engineering/design potential.
- Cheyenne Ziegler
- , Jonathan Martin
- & Faruck Morcos
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| Open AccessControlling protein stability with SULI, a highly sensitive tag for stabilization upon light induction
Tools for the spatiotemporal control of protein abundance are valuable in studying diverse complex biological processes. Here, authors engineered a protein tag which is stabilized upon light induction but which quickly degrades the protein of interest in the dark, demonstrating control of protein stability in yeast and zebrafish.
- Miaowei Mao
- , Yajie Qian
- & Yi Yang
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Article
| Open AccessBi-terminal fusion of intrinsically-disordered mussel foot protein fragments boosts mechanical strength for protein fibers
High-yield production of well-performing protein materials is challenging due to their high molecular weights and repetitive sequences. Here the authors develop a method to boost the strength of low molecular-weight protein materials by bi-terminal fusion of intrinsically-disordered mussel foot protein fragments, while achieving high yield.
- Jingyao Li
- , Bojing Jiang
- & Fuzhong Zhang
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Article
| Open AccessAutomated optimisation of solubility and conformational stability of antibodies and proteins
Antibodies find key applications in research, diagnostics, and therapeutics, but their development can be impeded by poor stability or solubility. Here the authors developed a computational strategy that enables antibody optimisation, without affecting functionality.
- Angelo Rosace
- , Anja Bennett
- & Pietro Sormanni
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| Open AccessDiscovering highly potent antimicrobial peptides with deep generative model HydrAMP
Antimicrobial peptides emerge as compounds that can alleviate the global health hazard of antimicrobial resistance. Here, the authors propose HydrAMP, an extended conditional variational autoencoder. HydrAMP generated antimicrobial peptides with high activity against bacteria, including multidrug-resistant species.
- Paulina Szymczak
- , Marcin Możejko
- & Ewa Szczurek
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| Open AccessAn engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration
Mitochondrial fatty acid synthesis (mtFAS) generates the precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. Here, the authors generated an engineered variant of human 2E-enoyl-ACP reductase (MECR) of mtFAS to study the role of long chain fatty acids.
- M. Tanvir Rahman
- , M. Kristian Koski
- & Kaija J. Autio
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| Open AccessThe RESP AI model accelerates the identification of tight-binding antibodies
High-affinity antibodies are often identified through directed evolution but deep leaning methods hold great promise. Here the authors report RESP, a pipeline for efficient identification of high affinity antibodies, and apply this to the PD-L1 antibody Atezolizumab.
- Jonathan Parkinson
- , Ryan Hard
- & Wei Wang
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Article
| Open AccessDesign and characterization of a protein fold switching network
In this work the authors investigate the structure-sequence dependance. The ability to design and characterize proteins at interfaces between three common folds suggests that fold switching is an intrinsic feature of protein folding language and likely important in the evolution of protein structure and function.
- Biao Ruan
- , Yanan He
- & Philip N. Bryan
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Article
| Open AccessDifferential sensing with arrays of de novo designed peptide assemblies
Differential sensing aims to mimic senses such as taste and smell through the use of synthetic receptors. Here, the authors show that arrays of de novo designed peptide assemblies can be used as sensor components to distinguish various analytes and complex mixtures.
- William M. Dawson
- , Kathryn L. Shelley
- & Derek N. Woolfson
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| Open AccessPrediction of designer-recombinases for DNA editing with generative deep learning
Design of recombinases with new target sites is usually achieved through cycles of directed molecular evolution. Here the authors report Recombinase Generator, RecGen, an algorithm for generation of designer-recombinases; they perform experimental validation to show that this can predict recombinase sequences.
- Lukas Theo Schmitt
- , Maciej Paszkowski-Rogacz
- & Frank Buchholz
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| Open AccessRational enzyme design for enabling biocatalytic Baldwin cyclization and asymmetric synthesis of chiral heterocycles
Chiral heterocyclic compounds are privileged structures in medicinal chemistry. Here, the authors report an in silico strategy for the enzymatic synthesis of pharmaceutically significant chiral N- and O-heterocycles via Baldwin cyclization of hydroxy- and amino-substituted epoxides and oxetanes using epoxide hydrolase mutants.
- Jun-Kuan Li
- , Ge Qu
- & Zhoutong Sun
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Article
| Open AccessOrthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase
Engineering enzymes to accept noncanonical cofactor biomimetics is difficult. Here, the authors establish a self-sufficient growth selection method and demonstrate its application in engineering the Lactobacillus pentosus NADH oxidase to efficiently recycle reduced nicotinamide mononucleotide (NMNH).
- Edward King
- , Sarah Maxel
- & Han Li
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| Open AccessSampling of structure and sequence space of small protein folds
In this work the authors provide a computational workflow for the parallel, from scratch, design of proteins to rapidly explore the shape diversity of protein folds.
- Thomas W. Linsky
- , Kyle Noble
- & Eva-Maria Strauch
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| Open AccessDesign of synthetic collagens that assemble into supramolecular banded fibers as a functional biomaterial testbed
Molecular level control is required to capture the folding and supramolecular assembly of collagen in mimetic materials. Here, the authors report on the creation of a synthetic collagen which assembles into banded fibers, recaptures structural properties of natural collagen and which can act as a testbed for design and experimentation
- Jinyuan Hu
- , Junhui Li
- & Fei Xu
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Article
| Open AccessDe novo design of immunoglobulin-like domains
The immunoglobulin domain framework of antibodies has been a long standing design challenge. Here, the authors describe design rules for tailoring these domains and show they can be accurately designed, de novo, with high stability and the ability to scaffold functional loops.
- Tamuka M. Chidyausiku
- , Soraia R. Mendes
- & Enrique Marcos
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| Open AccessSystematic profiling of conditional degron tag technologies for target validation studies
Conditional Degron Tags are a valuable tool to validate and study novel therapeutic targets. Here, the authors compared 5 orthogonal tags across 16 unique proteins and provide a panel of vectors for users to systematically screen the tags with their own protein of interest.
- Daniel P. Bondeson
- , Zachary Mullin-Bernstein
- & Alessandra Ianari
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| Open AccessSubstrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
Efficient engineering of enzymes for expanded substrate scope is currently challenging. Here, the authors develop simple principles of how to design and interpret Substrate Multiplexed Screening assays to guide protein engineering to enable activity improvements with simultaneously with multiple substrates.
- Allwin D. McDonald
- , Peyton M. Higgins
- & Andrew R. Buller
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Article
| Open AccessMVsim is a toolset for quantifying and designing multivalent interactions
Arising through multiple binding elements, multivalency can specify the avidity, duration, cooperativity, and selectivity of biomolecular interactions, but quantitative prediction and design of these properties has remained challenging. Here the authors enable facile analysis and engineering of multivalent binding by developing MVsim, a simulator that incorporates biochemical and biophysical parameters of interacting molecules and is accessible through a graphical user interface.
- Bence Bruncsics
- , Wesley J. Errington
- & Casim A. Sarkar
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| Open AccessDe novo protein design of photochemical reaction centers
De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build enzymes for efficient energy conversion. Here, the authors develop an artificial photosynthetic reaction center that functions without the need for sacrificial electron donors or acceptors.
- Nathan M. Ennist
- , Zhenyu Zhao
- & Christopher C. Moser
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| Open AccessEngineering an autonomous VH domain to modulate intracellular pathways and to interrogate the eIF4F complex
Approaches have been devised to increase the discovery rate of intrabodies but often these yield results that aren’t functional in cells. Here the authors engineer and optimise an autonomous and disulphide-free human VH domain for intracellular expression, and they identify several VH domain binders against eIF4E.
- Yuri Frosi
- , Yen-Chu Lin
- & Christopher J. Brown
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| Open AccessStructure-guided and phage-assisted evolution of a therapeutic anti-EGFR antibody to reverse acquired resistance
Acquired resistance to cetuximab can be mediated by generation of mutations in the EGFR ectodomain. Here the authors report a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse resistance without altering the binding epitope or undermining antibody efficacy.
- Xinlei Zhuang
- , Zhe Wang
- & Liqiang Pan
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| Open Accessα-Synuclein fibril-specific nanobody reduces prion-like α-synuclein spreading in mice
Butler et al. selected disulfide bond-free nanobodies to target α-synuclein fibrils. Nanobody PFFNB2 was shown to disaggregate α-synuclein fibrils in vitro and inhibit α-synuclein pathology development in neuron cultures and mouse models.
- Yemima R. Butler
- , Yuqing Liu
- & Wenjing Wang
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Article
| Open AccessTranscriptional programming in a Bacteroides consortium
Bacteroides species are prominent members of the human gut microbiota. Here the authors develop a platform technology (consortium transcriptional programming) in five human donor Bacteroides chassis cells—enabling complex decision-making within said community, which can be used for the development of living therapeutics.
- Brian D. Huang
- , Thomas M. Groseclose
- & Corey J. Wilson
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| Open AccessCo-optimization of therapeutic antibody affinity and specificity using machine learning models that generalize to novel mutational space
Optimising antibody properties such as affinity can be detrimental to other key properties. Here the authors use machine learning to simplify the identification of antibodies with co-optimal levels of affinity and specificity for a clinical-stage antibody that displays high levels of on- and off-target binding.
- Emily K. Makowski
- , Patrick C. Kinnunen
- & Peter M. Tessier
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| Open AccessSpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly
The SpyCatcher-SpyTag system allows protein anchoring and nanoassembly. Here, the authors engineer SpySwitch, a dually switchable Catcher which allows gentle purification of SpyTagged proteins prior to downstream applications such as the assembly of virus-like particles.
- Susan K. Vester
- , Rolle Rahikainen
- & Mark Howarth
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Article
| Open AccessComputationally designed hyperactive Cas9 enzymes
The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. Here, the authors use computational design to discover Cas9 enzymes with increased activity.
- Pascal D. Vos
- , Giulia Rossetti
- & Oliver Rackham
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| Open AccessA topological refactoring design strategy yields highly stable granulopoietic proteins
Skokowa et al. reconstruct the fold of a granulopoietic cytokine, resulting in de novo, hyperstable, highly active proteins with therapeutic potential for treating several neutropenia disorders.
- Julia Skokowa
- , Birte Hernandez Alvarez
- & Mohammad ElGamacy
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| Open AccessA high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors
Fluorescent biosensors are important tools for studying cellular metabolism, but development and optimization are challenging. Koveal et al. present a high-throughput multiparameter screen for sensor performance, and used it to generate LiLac, a high-performance, quantitative lactate sensor.
- Dorothy Koveal
- , Paul C. Rosen
- & Gary Yellen
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Article
| Open AccessSynthetic cells with self-activating optogenetic proteins communicate with natural cells
Synthetic biology and engineering approaches are harnessed to incorporate new capabilities in synthetic cells. Here, the authors designed bioluminescent signaling mechanisms for intracellular and intercellular synthetic-to-natural cell communication.
- Omer Adir
- , Mia R. Albalak
- & Avi Schroeder
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| Open AccessMetal-responsive regulation of enzyme catalysis using genetically encoded chemical switches
Dynamic control over protein function is a central challenge in synthetic biology. Here the authors present an integrated computational and experimental workflow for engineering reversible protein switches; metal-chelating unnatural amino acids genetically encoded into two conformationally dynamic enzymes to yield robust switches.
- Yasmine S. Zubi
- , Kosuke Seki
- & Jared C. Lewis
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| Open AccessStructure-based design of stabilized recombinant influenza neuraminidase tetramers
Influenza virus neuraminidase (NA) is a drug target and a potential vaccine antigen. Here, the authors provide a detailed analysis of the conformational stability of NA, and show how expression and stability of recombinant NA antigens can be strengthened through structure-based design.
- Daniel Ellis
- , Julia Lederhofer
- & Masaru Kanekiyo
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| Open AccessCryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation
Lateral opening of the LptDE transporter in the outer membrane of Gram-negative bacteria is necessary for insertion of lipopolysaccharides. Here, Botte et al. report a cryo-EM structure of a partially opened LptDE transporter, in complex with rigid chaperones derived from nanobodies.
- Mathieu Botte
- , Dongchun Ni
- & Michael Hennig
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Article
| Open AccessSelective optogenetic control of Gq signaling using human Neuropsin
Gq proteins are one of four major classes of G proteins; optogenetic receptors for selective and repetitive activation of Gq proteins with fast kinetics are lacking. Here the authors report UV light-dependent Gq signalling using human Neuropsin (hOPN5) and demonstrate its potential as an optogenetic tool.
- Ahmed Wagdi
- , Daniela Malan
- & Tobias Bruegmann
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Article
| Open AccessDe novo design and directed folding of disulfide-bridged peptide heterodimers
Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Here the authors report de novo design and directed folding of peptide heterodimers crosslinked through multiple disulfide bonds, which can be explored as chemical tools for orthogonal labeling of proteins and preparing protein hybrids.
- Sicong Yao
- , Adam Moyer
- & Chuanliu Wu
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| Open AccessProtein sequence design with a learned potential
Rational protein design to achieve a given protein backbone conformation is needed to engineer specific functions. Here Anand et al. describe a machine learning method using a learned neural network potential for fixed-backbone protein design.
- Namrata Anand
- , Raphael Eguchi
- & Po-Ssu Huang
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| Open AccessAlgorithm-aided engineering of aliphatic halogenase WelO5* for the asymmetric late-stage functionalization of soraphens
The late-stage functionalization of unactivated carbon–hydrogen bonds is a difficult but important task, which has been met with promising but limited success through synthetic organic chemistry. Here the authors use machine learning to engineer WelO5* halogenase variants, which led to regioselective chlorination of inert C–H bonds on a representative polyketide that is a non-natural substrate for the enzyme.
- Johannes Büchler
- , Sumire Honda Malca
- & Rebecca Buller
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| Open AccessDesign principles for site-selective hydroxylation by a Rieske oxygenase
SxtT and GxtA are Rieske oxygenases that are involved in paralytic shellfish toxin biosynthesis and catalyze monohydroxylation reactions at different positions on the toxin scaffold. Here, the authors present crystal structures of SxtT and GxtA with the native substrates β-saxitoxinol and saxitoxin as well as a Xenon-pressurized structure of GxtA, which reveal a substrate access tunnel to the active site. Through structure-based mutagenesis studies the authors identify six residues in three different protein regions that determine the substrate specificity and site selectivity of SxtT and GxtA. These findings will aid the rational engineering of other Rieske oxygenases.
- Jianxin Liu
- , Jiayi Tian
- & Jennifer Bridwell-Rabb
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Article
| Open AccessHelical structure motifs made searchable for functional peptide design
Here, we present TP-DB; a pattern-based search engine based on 1.67 million helices from the Protein Database (PDB). We demonstrate the utility of TP-DB in identifying microbe-specific antigens, as well as the design of antimicrobial peptides and Protein-protein interaction blockers.
- Cheng-Yu Tsai
- , Emmanuel Oluwatobi Salawu
- & Lee-Wei Yang
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| Open AccessMechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography
Shark antibodies (Variable New Antigen Receptors, VNARs) are the smallest naturally occurring antibody fragments. Here, the authors screen a VNAR phage display library against the SARS-CoV2 receptor binding domain (RBD) and identify VNARs that neutralize the SARSCoV-2 virus and discuss their mechanisms of viral neutralization.
- Obinna C. Ubah
- , Eric W. Lake
- & Caroline J. Barelle
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Article
| Open AccessProbing ion channel functional architecture and domain recombination compatibility by massively parallel domain insertion profiling
Here, the authors perform a large-scale, high-throughput biochemical assay to determine the compatibility of over 300,000 domain recombination variants of the inward rectifier K+ channel Kir2.1. They derive rules for designing domain insertion variants that fold and traffic to the cell surface and conclude that the insertion of domains at protein termini is evolutionary favoured.
- Willow Coyote-Maestas
- , David Nedrud
- & Daniel Schmidt
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Article
| Open AccessA genetically encoded fluorescent biosensor for extracellular l-lactate
l-lactate is an important intercellular energy currency. Here the authors report a genetically encoded biosensor eLACCO1.1 to monitor extracellular l-lactate; they use eLACCO1.1 to image extracellular l-lactate in cultured mammalian cells and brain tissue.
- Yusuke Nasu
- , Ciaran Murphy-Royal
- & Robert E. Campbell
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Article
| Open AccessDirected evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex
Facilitated dissociation is a mechanism where antibody-mediated disruption of high-affinity complexes can enhance the therapeutic effects of a drug. Here the authors present a yeast display approach to select and engineer omalizumab variants that dissociate receptor-bound IgE to accelerate its inhibition of the allergic response.
- Luke F. Pennington
- , Pascal Gasser
- & Theodore S. Jardetzky
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Article
| Open AccessVersatile selective evolutionary pressure using synthetic defect in universal metabolism
Rational design of enzymes with new or improved properties is rarely straightforward, and artificial selection pressure approaches that link an improvement in the target to cell growth are an alternative. Here, the authors show that diverse enzymes sharing the ubiquitous cofactor NAD(P)+ can substitute for defective NAD+ regeneration, representing a very broadly-applicable artificial selection.
- Lara Sellés Vidal
- , James W. Murray
- & John T. Heap
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Article
| Open AccessMechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design
Certain bovine antibodies have ultra-long long complementarity-determining regions (CDRs) that contain a knob for antigen interaction, which is connected to the antibody through a stalk. Here, the authors combine biophysical experiments and MD simulations and show that the stalk length is critical for the folding and stability of these antibodies. The authors also demonstrate that ultra-long bovine CDRs can be grafted into human antibodies, and furthermore show that de novo designed mini-domains that bind to the SARS-CoV-2 spike protein with high affinity can be integrated as a knob in ultra-long CDRs in bovine and human antibodies, which neutralize SARS-CoV-2.
- Hristo L. Svilenov
- , Julia Sacherl
- & Johannes Buchner
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| Open AccessDisulfide-compatible phage-assisted continuous evolution in the periplasmic space
The directed evolution of antibodies yields important tools for research and therapy. Here the authors develop a periplasmic phage-assisted continuous evolution platform for improvement of protein-protein interactions in the disulfidecompatible E. coli periplasm.
- Mary S. Morrison
- , Tina Wang
- & David R. Liu
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Article
| Open AccessEfficient generative modeling of protein sequences using simple autoregressive models
Deep learning is a powerful tool for the design of novel protein sequences, yet can be computationally very inefficient. Here the authors propose using simple forecasting models to efficiently generate a large number of novel protein structures.
- Jeanne Trinquier
- , Guido Uguzzoni
- & Martin Weigt
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Article
| Open AccessA rational blueprint for the design of chemically-controlled protein switches
Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions into OFF- and ON-switches active in cells.
- Sailan Shui
- , Pablo Gainza
- & Bruno E. Correia
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| Open AccessECNet is an evolutionary context-integrated deep learning framework for protein engineering
Protein engineering is an active area of research in which machine learning has proven quite powerful. Here, the authors present a deep learning method that integrates both general and protein-specific sequence representations to improve the engineering of one’s protein of interest.
- Yunan Luo
- , Guangde Jiang
- & Jian Peng
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Article
| Open AccessComprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
Proteins evolve through the modular rearrangement of domains. Here the authors introduce MISER, a minimization by iterative size-exclusion and recombination method to make all possible deletions of a protein, uncovering functions for Cas9 domains involved in DNA binding.
- Arik Shams
- , Sean A. Higgins
- & David F. Savage