Development of potent in vivo mutagenesis plasmids with broad mutational spectra

Badran, Ahmed H.; Liu, David R.

doi:10.1038/ncomms9425

Cited by 161 publications

(203 citation statements)

References 36 publications

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“…This design imposes selection pressure to recognize many different PAM sequences, as well as to maintain compatibility with different target DNA sequences. All three AP libraries were introduced into host E. coli cells harboring the mutagenesis plasmid MP6 17 . The resulting host cells were incubated overnight with SP containing ω(I12N)–dCas9.…”

Section: Resultsmentioning

confidence: 99%

“…During PACE, host E. coli cells continuously dilute an evolving population of bacteriophages (selection phage, SP). Since dilution occurs faster than cell division but slower than phage replication, only the SP, and not the host cells, can accumulate mutations 17 . Each SP carries a gene to be evolved instead of a phage gene (gene III) that is required for the production of infectious progeny phage.…”

mentioning

confidence: 99%

“…1a). As phage replication can occur in as little as 10 minutes, PACE enables hundreds of generations of directed evolution to occur per week without researcher intervention 17–22 .…”

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confidence: 99%

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Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Miller

Geurts

et al. 2018

Nature

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Summary A key limitation to the use of CRISPR-Cas9 proteins for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), this PAM requirement is NGG. No natural or engineered Cas9 variants shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA, and GAT. The PAM compatibility of xCas9 is the broadest reported to date among Cas9s active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and both cytidine and adenine base editing. Remarkably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Miller

Geurts

et al. 2018

Nature

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“…(4) Protein characterization: the target protein's activity needs to be analyzed with a suitable reporter assay. not include a random mutagenesis plasmid 35 . This means that structural information or a partial understanding of how a set of amino acid changes will affect the target protein's activity is required to run our system.…”

Section: Comparison With Other Methodsmentioning

confidence: 99%

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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“…Our TEMrev-GFPrev reporter system should be of use to fine-tune LF-Pol I-expressing cells and other existing mutator strain ssuch as XL-1 red (42), the MP6 mutagenesis system (43), or strains with altered dNTP pools (40) to identify conditions supporting constant mutation rates over time.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

Standley

Allen

Cervantes

et al. 2017

Methods in Enzymology

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Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. The present article focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor-intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations.

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Development of potent in vivo mutagenesis plasmids with broad mutational spectra

Cited by 161 publications

References 36 publications

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

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

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

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