Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance

Badran, Ahmed H.; Guzov, Victor M.; Huai, Qing; Kemp, Melissa M.; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M.; Evdokimov, A.G.; Moshiri, Farhad; Turner, Keith H.; Wang, Ping; Malvar, Thomas; Liu, David R.

doi:10.1038/nature17938

Cited by 177 publications

(220 citation statements)

References 41 publications

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“…All mammalian cell and bacterial plasmids generated in this work were assembled using the USER cloning method as previously described 39 and starting material gene templates were synthetically accessed as either bacterial or mammalian codon-optimized gBlock Gene Fragments (Integrated DNA Technologies). All sgRNA expression plasmids were constructed by a 1-piece blunt-end ligation of a PCR product containing a variable 20-nt sequence corresponding to the desired sgRNA targeted site.…”

Section: Methodsmentioning

confidence: 99%

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Gaudelli

Komor

Rees

et al. 2017

Nature

Self Cite

3,214

2,886

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Summary The spontaneous deamination of cytosine is a major source of C•G to T•A transitions, which account for half of known human pathogenic point mutations. The ability to efficiently convert target A•T base pairs to G•C therefore could advance the study and treatment of genetic diseases. While the deamination of adenine yields inosine, which is treated as guanine by polymerases, no enzymes are known to deaminate adenine in DNA. Here we report adenine base editors (ABEs) that mediate conversion of A•T to G•C in genomic DNA. We evolved a tRNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs (e.g., ABE7.10), that convert target A•T to G•C base pairs efficiently (~50% in human cells) with very high product purity (typically ≥ 99.9%) and very low rates of indels (typically ≤ 0.1%). ABEs introduce point mutations more efficiently and cleanly than a current Cas9 nuclease-based method, induce less off-target genome modification than Cas9, and can install disease-correcting or disease-suppressing mutations in human cells. Together with our previous base editors, ABEs advance genome editing by enabling the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

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Section: Methodsmentioning

confidence: 99%

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Gaudelli

Komor

Rees

et al. 2017

Nature

Self Cite

3,214

2,886

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“…The design of randomized oligonucleotides for overlap PCR is similar to conventional Gibson primer design 38 , and the RNA polymerase ω subunit (RpoZ; yellow) is fused to the evolving protein (red). Target protein binding of the evolving protein 24 enables the localization of RNA polymerase upstream of gene VI, initiating gene expression from the P lacZ-opt promoter. (e) The T7 polymerase (gray) is inhibited when bound to T7 lysozyme (dark blue), as it inhibits transcription initiation and the transition from initiation to elongation 44 .…”

Section: Experimental Designmentioning

confidence: 99%

“…Proteolysis of the target cleavage site (red) by an evolving protease 23 activates the T7 RNA polymerase, enabling gene VI expression downstream of the T7 promoter. Gene VI is annotated with an asterisk where originally conditional gene III was used instead of gene VI with PACE 10,[22][23][24] . Gene III and gene VI are both minor coat proteins, each present in three to five copies per phage particle 21 .…”

Section: Experimental Designmentioning

confidence: 99%

“…4). With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been proposed (e.g., biosensors 25 or hybrids with chemical evolution 26 ) can be adapted to this method if the target protein's activity can be linked to conditional M13 phage production.…”

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confidence: 99%

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Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

2017

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Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. these include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. a typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (pM) library expressing variants of the gene of interest (GoI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. the system has been developed for the selection of new orthogonal transcription factors (tFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage l cI tF against two synthetic bidirectional promoters. the evolved tF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. this protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. the chances of propagating nonfunctional library members because of multiple infections. A GOI with the desired characteristics upregulates gene VI expression on the AP, completing the phage life cycle. For example, a randomized TF library member that activates an artificial promoter upstream of gVI will increase its own phage production (Fig. 4a). In this way, a protein with novel desired properties can be selected after several rounds of reinfection. Applications of the methodThe method has been used to evolve a set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants, and multi-input promoter architectures, and these switches have been successfully applied in downstream synthetic gene circuits 19 . In general, the method is capable of evolving any gene-or gene circuit function-on the PM that can be linked to pVI production. This is analogous to previous uses of phageassisted continuous evolution (PACE) 22 (Fig. 4). With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been...

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“…One advantage of transforming crops with genes for natural antimicrobial substances is that one can employ in-vitro techniques of molecular evolution to broaden the range of molecular targets of such antimicrobials [77]. Such techniques potentially can be employed to reverse the buildup of pathogen resistance to the antimicrobial.…”

Section: Producing Antimicrobial Compoundsmentioning

confidence: 99%

Genetic Engineering and Sustainable Crop Disease Management: Opportunities for Case-by-Case Decision-Making

Vincelli

2016

Sustainability

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Genetic engineering (GE) offers an expanding array of strategies for enhancing disease resistance of crop plants in sustainable ways, including the potential for reduced pesticide usage. Certain GE applications involve transgenesis, in some cases creating a metabolic pathway novel to the GE crop. In other cases, only cisgenessis is employed. In yet other cases, engineered genetic changes can be so minimal as to be indistinguishable from natural mutations. Thus, GE crops vary substantially and should be evaluated for risks, benefits, and social considerations on a case-by-case basis. Deployment of GE traits should be with an eye towards long-term sustainability; several options are discussed. Selected risks and concerns of GE are also considered, along with genome editing, a technology that greatly expands the capacity of molecular biologists to make more precise and targeted genetic edits. While GE is merely a suite of tools to supplement other breeding techniques, if wisely used, certain GE tools and applications can contribute to sustainability goals.

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Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance

Cited by 177 publications

References 41 publications

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

Genetic Engineering and Sustainable Crop Disease Management: Opportunities for Case-by-Case Decision-Making

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