Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells

Erdoğan, Mutlu; Fabritius, Arne; Basquin, J.; Griesbeck, Oliver

doi:10.1016/j.chembiol.2020.02.004

Cited by 30 publications

(57 citation statements)

References 57 publications

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“…The G-quartets stabilize rigid chromophore conformations by stacking interactions that largely govern the photophysical properties 34 – 36 . Unlike the near-planar fluorinated ligands DFHBI and DFHO in Spinach and Corn 7 , 20 , 21 , the fluorophores DMHBO + and DMHBI + in Chili adopt moderately twisted conformations, resembling those found in some LSS fluorescent proteins such as LSSmKate1 19 and mCRISPred 37 (Supplementary Fig. 20 and Supplementary Table 3 ).…”

Section: Discussionmentioning

confidence: 94%

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

et al. 2021

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Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.

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Section: Discussionmentioning

confidence: 94%

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

et al. 2021

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“…[65][66][67][68] These approaches, however, require up-front development of a template sequence carrying the desired mutation of interest. Although useful for validation studies, targeted mutations, and singlesite saturation, 69 it remains difficult to envision the successful use of Figure 6. Overview of Currently Available Approaches for CRISPR-Directed Evolution Five classes of CRISPR-based genome editing systems are highlighted.…”

Section: Discussionmentioning

confidence: 99%

enAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9

et al. 2021

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While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug's target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.

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“…This makes it difficult to compare and quantify brightness across mutants without re-cloning them behind the original RBS. Emerging strategies for directed evolution in mammalian cells are potential solutions to this limitation [15,16]. Spontaneous mutagenesis in continuous style may further boost its screening efficiency with high throughput and minimal human intervention [17].…”

Section: Discussionmentioning

confidence: 99%

Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

2020

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The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.

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Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells

Cited by 30 publications

References 57 publications

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

enAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9

Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

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