
단백질-단백질 상호작용 또는 근접성을 이용하여 CRISPR-Cas9 가이드 RNA 복합체 형성을 유도함으로써 유전체 편집을 제어할 수 있는 새로운 전략을 제시한다.


coremsg

One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here we demonstrate a strategy termed “P3 editing”, which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically-induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.

### Competing Interest Statement

The University of Washington has filed a patent application based on this work, in which J.C., W.C., and J.S. are listed as inventors. J.S. is a scientific advisory board member, consultant, and/or co-founder of Cajal Neuroscience, Guardant Health, Maze Therapeutics, Camp4 Therapeutics, Phase Genomics, Adaptive Biotechnologies, Scale Biosciences, Sixth Street Capital, Pacific Biosciences, and Prime Medicine. The remaining authors declare no competing interests.

A molecular proximity sensor based on an engineered, dual-component guide RNA

단백질-단백질-근접성에-기반한-유전체-편집-센서-개발


단백질-단백질 근접성에 기반한 유전체 편집 센서 개발


title_rewrite


A molecular proximity sensor based on an engineered, dual-component guide RNA can link specific protein-protein interactions or chemically-induced dimerization to the activation of CRISPR-based genome editing tools, enabling programmable control of genome editing outcomes.


a-molecular-proximity-sensor-for-programmable-genome-editing-via-engineered-guide-rna


A Molecular Proximity Sensor for Programmable Genome Editing via Engineered Guide RNA



The bacterial retron system Eco2 can be used to effectively express and characterize functional DNA aptamers, such as the fluorogenic Lettuce aptamer, within living cells.


DNA aptamers are short, single-stranded DNA molecules that bind specifically to a range of targets such as proteins, cells, and small molecules. Typically, they are utilized in the development of therapeutic agents, diagnostics, drug delivery systems, and biosensors. Although aptamers perform well in controlled extracellular environments, their intracellular use has been less explored due to challenges of expressing them in vivo. In this study, we employed the bacterial retron system Eco2, to express a DNA light-up aptamer in Escherichia coli . Both in vitro and in vivo assays confirm that structure-guided insertion of the aptamer domain into the non-coding region of the retron enables reverse transcription and folding of functional aptamer constructs in vivo. Notably, we find only a limited correlation between in vitro and in vivo aptamer performance, suggesting marked folding differences between the two environments. Our findings demonstrate that retrons can be used to effectively express short DNA aptamers within living cells, potentially broadening and optimizing their application in intracellular settings.

### Competing Interest Statement

The authors have declared no competing interest.

Intracellular Expression of a Fluorogenic DNA Aptamer Using Retron Eco2

intracellular-expression-and-characterization-of-a-fluorogenic-dna-aptamer-using-the-bacterial-retron-eco2-system


Intracellular Expression and Characterization of a Fluorogenic DNA Aptamer Using the Bacterial Retron Eco2 System



Engineered RNA-sensing CRISPR guide RNAs (iSBH-sgRNAs) enable conditional activation of CRISPR transcriptional regulators in response to specific RNA sequences in mammalian cells and zebrafish embryos.


Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest while preventing unwanted activity in other cells. Here, we present a simple and specific platform for modulating CRISPR activity in response to RNA detection through engineering Streptococcus pyogenes Cas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. The engineered sgRNAs become activated upon recognising complementary RNAs, thus enabling Cas9 to perform its function. Our approach enables CRISPR activation in response to RNA detection in both HEK293T cells and zebrafish embryos. Iterative design optimisations allowed the development of computational tools for generating sgRNAs capable of detecting RNA sequences of choice. Mechanistic investigations reveal that engineered sgRNAs are cleaved during RNA detection, and we identify key positions that benefit from chemical modifications to improve the stability of engineered sgRNAs in vivo . Our sensors open up novel opportunities for developing new research and therapeutic applications using CRISPR activation in response to endogenous RNA biomarkers.

### Competing Interest Statement

The authors have declared no competing interest.

Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

modular-crispr-transcriptional-activators-enabled-by-rna-sensing-guide-rnas-in-mammalian-cells-and-zebrafish-embryos


Modular CRISPR Transcriptional Activators Enabled by RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos



Integrating an optogenetic module into a repressilator circuit enables the use of light to synchronize, entrain, and detune oscillations in gene expression within single cells or entire populations.


Synthetic genetic oscillators can serve as internal clocks within engineered cells to program periodic expression. However, cell-to-cell variability introduces a dispersion in the characteristics of these clocks that drives the population to complete desynchronization. Here we introduce the optorepressilator, an optically controllable genetic clock that combines the repressilator, a three-node synthetic network in E. coli , with an optogenetic module enabling to reset, delay, or advance its phase using optical inputs. We demonstrate that a population of optorepressilators can be synchronized by transient green light exposure or entrained to oscillate indefinitely by a train of short pulses, through a mechanism reminiscent of natural circadian clocks. Furthermore, we investigate the system’s response to detuned external stimuli observing multiple regimes of global synchronization. Integrating experiments and mathematical modeling, we show that the entrainment mechanism is robust and can be understood quantitatively from single cell to population level.

### Competing Interest Statement

The authors have declared no competing interest.

Light-driven synchronization of optogenetic clocks

optogenetic-synchronization-and-entrainment-of-a-synthetic-genetic-oscillator


Optogenetic Synchronization and Entrainment of a Synthetic Genetic Oscillator



새로 설계된 미니바인더는 다양한 합성 수용체에서 항원 센서로 기능할 수 있으며, 이를 통해 세포 공학 도구의 감지 범위를 크게 확장할 수 있다.


Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled “smart” therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo -designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.

### Competing Interest Statement

HE-S is an employee of Altos Labs

De novo-designed minibinders expand the synthetic biology sensing repertoire

합성-생물학-센싱-범위를-확장하는-새로운-설계-미니바인더


합성 생물학 센싱 범위를 확장하는 새로운 설계 미니바인더



デノボ設計ミニバインダーは、様々な合成受容体のアンチゲンセンサーとして機能し、細胞工学ツールの感知能力を大幅に拡張する可能性がある。


合成生物学的センサーレパートリーを拡張するためのデノボ設計ミニバインダー


De novo-designed minibinders can be readily adapted as antigen sensors across diverse synthetic receptor platforms, including proteolytic receptors and chimeric antigen receptors, enabling expanded sensing capabilities for engineered cell therapies.


de-novo-designed-minibinders-expand-the-synthetic-biology-sensing-repertoire-for-engineered-cell-therapies


De Novo-Designed Minibinders Expand the Synthetic Biology Sensing Repertoire for Engineered Cell Therapies
