Faculty Opinions recommendation of Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors.

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

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Studying the function of methyltransferases using nucleotide analogue based random mutagenesis

Collection Symposium Series ◽

10.1135/css199902209 ◽

1999 ◽

Author(s):

Victoria H. Harris ◽

Alan Hamilton ◽

David M. Williams ◽

David P. Hornby

Keyword(s):

Random Mutagenesis ◽

Nucleotide Analogue

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Tc8, a Tourist-like Transposon in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/158.3.1081 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1081-1088 ◽

Cited By ~ 2

Author(s):

Quang Hien Le ◽

Kime Turcotte ◽

Thomas Bureau

Keyword(s):

Caenorhabditis Elegans ◽

Expressed Sequence Tags ◽

Large Family ◽

Insertion Sequences ◽

Normal Plant ◽

Nematode Caenorhabditis Elegans ◽

Plant Genomes ◽

Plant Genes ◽

Expressed Sequence ◽

Eukaryotic Genomes

Abstract Members of the Tourist family of miniature inverted-repeat transposable elements (MITEs) are very abundant among a wide variety of plants, are frequently found associated with normal plant genes, and thus are thought to be important players in the organization and evolution of plant genomes. In Arabidopsis, the recent discovery of a Tourist member harboring a putative transposase has shed new light on the mobility and evolution of MITEs. Here, we analyze a family of Tourist transposons endogenous to the genome of the nematode Caenorhabditis elegans (Bristol N2). One member of this large family is 7568 bp in length, harbors an ORF similar to the putative Tourist transposase from Arabidopsis, and is related to the IS5 family of bacterial insertion sequences (IS). Using database searches, we found expressed sequence tags (ESTs) similar to the putative Tourist transposases in plants, insects, and vertebrates. Taken together, our data suggest that Tourist-like and IS5-like transposons form a superfamily of potentially active elements ubiquitous to prokaryotic and eukaryotic genomes.

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Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity

Nature Communications ◽

10.1038/s41467-021-22519-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jianan Li ◽

Wenxia Yu ◽

Shisheng Huang ◽

Susu Wu ◽

Liping Li ◽

...

Keyword(s):

Rna Editing ◽

Potential Interaction ◽

Reporter System ◽

Independent Manner ◽

Robust Detection ◽

Guide Rna ◽

Highly Efficient ◽

Adenine Base

AbstractBoth adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.

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Combining random mutagenesis, structure-guided design and next-generation sequencing to mitigate polyreactivity of an anti-IL-21R antibody

mAbs ◽

10.1080/19420862.2021.1883239 ◽

2021 ◽

Vol 13 (1) ◽

pp. 1883239

Author(s):

Sharon M. Campbell ◽

Joseph DeBartolo ◽

James R. Apgar ◽

Lydia Mosyak ◽

Virginie McManus ◽

...

Keyword(s):

Next Generation Sequencing ◽

Random Mutagenesis ◽

Next Generation ◽

Generation Sequencing

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Random mutagenesis to identify novelHelicobacter mustelaevirulence factors

FEMS Immunology & Medical Microbiology ◽

10.1111/j.1574-695x.2006.00207.x ◽

2007 ◽

Vol 50 (2) ◽

pp. 257-263 ◽

Cited By ~ 8

Author(s):

Tadhg Ó Cróinín ◽

Aisling McCormack ◽

Arnoud H.M. van Vliet ◽

Johannes G. Kusters ◽

Billy Bourke

Keyword(s):

Random Mutagenesis

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Evolutionary design of optimal surface topographies for biomaterials

Scientific Reports ◽

10.1038/s41598-020-78777-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Aliaksei Vasilevich ◽

Aurélie Carlier ◽

David A. Winkler ◽

Shantanu Singh ◽

Jan de Boer

Keyword(s):

High Throughput Screening ◽

Random Mutagenesis ◽

Material Design ◽

Fitness Assessment ◽

Surface Topographies ◽

Optimal Surface ◽

Design Production ◽

Brute Force Approach ◽

Survival Of The Fittest ◽

Selection Of

AbstractNatural evolution tackles optimization by producing many genetic variants and exposing these variants to selective pressure, resulting in the survival of the fittest. We use high throughput screening of large libraries of materials with differing surface topographies to probe the interactions of implantable device coatings with cells and tissues. However, the vast size of possible parameter design space precludes a brute force approach to screening all topographical possibilities. Here, we took inspiration from Nature to optimize materials surface topographies using evolutionary algorithms. We show that successive cycles of material design, production, fitness assessment, selection, and mutation results in optimization of biomaterials designs. Starting from a small selection of topographically designed surfaces that upregulate expression of an osteogenic marker, we used genetic crossover and random mutagenesis to generate new generations of topographies.

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