StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins

Jacobs, C.; Badiee, Ryan K; Lin, Michael Z.

doi:10.1038/s41592-018-0041-z

Cited by 46 publications

(44 citation statements)

References 28 publications

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“…Using this system, they demonstrated dose-dependent transcription activation, but the system was irreversible. 88 In a similar approach, Tague et al used the NS3 protease domain and its inhibitor BILN-2061 as a ligand-inducible connection (LInC) to control the association of DNA binding and transcription activation domains. In their design, the viral protease was incorporated into dCas9-VPR such that the protease was positioned between the DNA binding sca ffold and the C-terminal region that contained a nuclear localization sequence (NLS) and the VPR transcription activation domain (dCas9-NS3-NLS-VPR).…”

Section: Control By Small-molecule Activatorsmentioning

confidence: 99%

Precision Control of CRISPR-Cas9 Using Small Molecules and Light

et al. 2019

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The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is an adaptive immune system of bacteria that has furnished several RNA-guided DNA endonucleases (e.g., Cas9) that are revolutionizing the field of genome engineering. Cas9 is being used to effect genomic alterations as well as in gene drives, where a particular trait may be propagated through a targeted species population over several generations. The ease of targeting catalytically impaired Cas9 to any genomic loci has led to development of technologies for base editing, chromatin imaging and modeling, epigenetic editing, and gene regulation. Unsurprisingly, Cas9 is being developed for numerous applications in biotechnology and biomedical research and as a gene therapy agent for multiple pathologies. There is a need for precise control of Cas9 activity over several dimensions, including those of dose, time, and space in these applications. Such precision controls, which are required of therapeutic agents, are particularly important for Cas9 as off-target effects, chromosomal translocations, immunogenic response, genotoxicity, and embryonic mosaicism are observed at elevated levels and with prolonged activity of Cas9. Here, we provide a perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas9.

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Section: Control By Small-molecule Activatorsmentioning

confidence: 99%

Precision Control of CRISPR-Cas9 Using Small Molecules and Light

et al. 2019

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“…The availability of ANR to basally localize a protein activity fused to NS3a away from a site of action, or to act as an intramolecular regulatory switch, provides an appealing option for reducing background, but protease-based NS3a control systems offer the possibility of obtaining the complete absence of a protein activity in the cell prior to the addition of a small molecule input. 10,11,27,42 However, the reduced background that can be obtained with protease-based systems comes at the expense of speed, as protein accumulation is required for activation. Furthermore, we have found that catalytically-dead NS3a is an equally effective component of PROCISiR as the active protease, which is advantageous for applications in which off-target proteolytic activity may be a concern.…”

Section: Discussionmentioning

confidence: 99%

Multi-input chemical control of protein dimerization for programming graded cellular responses

et al. 2019

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Chemical and optogenetic methods for post-translationally controlling protein function have enabled new discoveries and the engineering of synthetic cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of posttranslational behaviors that can be programmed we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs with engineered reader proteins that provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program diverse cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our receiver protein and suite Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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“…small molecule assisted shutoff or SMASh-tag; 78 ] could be useful for chasing old and new histones in parallel. Specifically, it will be interesting to determine if recently described orthogonal SMASh-tags [called stabilizable polypeptide linkers or StaPLs; 79 ] could be applied to studying histone dynamics. For example, by tagging two alleles of H3F3B with orthogonal StaPLs and epitope tags, it might be possible to distinguish old H3.3 (degron inhibited before switch and activated after switch) from new H3.3 (degron activated before switch and inhibited after switch).…”

Section: Discussionmentioning

confidence: 99%

Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells

et al. 2020

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In eukaryotes, nucleosomes form a barrier to DNA templated reactions and must be dynamically disrupted to provide access to the genome. During nucleosome (re)assembly, histones can be replaced by new histones, erasing post-translational modifications. Measuring histone turnover in mammalian cells has mostly relied on inducible overexpression of histones, which may influence and distort natural histone deposition rates. We have previously used recombination-induced tag exchange (RITE) to study histone dynamics in budding yeast. RITE is a method to follow protein turnover by genetic switching of epitope tags using Cre recombinase and does not rely on inducible overexpression. Here, we applied RITE to study the dynamics of the replicationindependent histone variant H3.3 in human cells. Epitope tag-switching could be readily detected upon induction of Cre-recombinase, enabling the monitoring old and new H3.3 in the same pool of cells. However, the rate of tag-switching was lower than in yeast cells. Analysis of histone H3.3 incorporation by chromatin immunoprecipitation did not recapitulate previously reported aspects of H3.3 dynamics such as high turnover rates in active promoters and enhancers. We hypothesize that asynchronous Cre-mediated DNA recombination in the cell population leads to a low time resolution of the H3.3-RITE system in human cells. We conclude that RITE enables the detection of old and new proteins in human cells and that the timescale of tag-switching prevents the capture of high turnover events in a population of cells. Instead, RITE might be more suited for tracking long-lived histone proteins in human cells.

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StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins

Cited by 46 publications

References 28 publications

Precision Control of CRISPR-Cas9 Using Small Molecules and Light

Precision Control of CRISPR-Cas9 Using Small Molecules and Light

Multi-input chemical control of protein dimerization for programming graded cellular responses

Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells

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