Genome editor-directed in vivo library diversification

Cheng, Cristina; Zhou, Minjie; Su, Qiwen; Steigmeyer, Alexandra; Niu, Jia

doi:10.1016/j.chembiol.2021.05.008

Cited by 9 publications

(6 citation statements)

References 55 publications

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“…Molecular evolution is the most commonly used and successful strategy to improve the performance of genome-editing tools. 48 However, mutagenic library-based screening is difficult in eukaryotic cells, especially in mammalian cells. As Cas9 is super-active in prokaryotic cells, it is difficult to filter out mutations with limited activity enhancement.…”

Section: Discussionmentioning

confidence: 99%

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

et al. 2023

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Section: Discussionmentioning

confidence: 99%

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

et al. 2023

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“…Another important aspect of library expression is the need to express exactly one single indicator variant per cell, thereby ensuring proper phenotype–genotype linkage. This can be achieved by several methods, including sparse transfection/transduction, recombination of a promoterless plasmid into an engineered genomic locus (landing pad) (Shukla et al., 2021), or in vivo library generation approaches (Cheng et al., 2021; Molina et al., 2022). While techniques involving sparse transfection or transduction are compatible with large libraries, they suffer from the limitations of transient expression (limiting the time for screening/selection) or random genomic integration (affecting GOI transcription levels), respectively.…”

Section: Future Directions In Indicator Engineeringmentioning

confidence: 99%

Engineering, applications, and future perspectives of GPCR‐based genetically encoded fluorescent indicators for neuromodulators

Rohner,

Lamothe‐Molina,

Patriarchi

2024

Journal of Neurochemistry

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This review explores the evolving landscape of G‐protein‐coupled receptor (GPCR)‐based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand‐binding properties, structural insights, and spectral diversity.

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“…Random mutagenesis makes rapid biomolecular engineering possible. CRISPR-based BEs allow edition at the target sites which were hybridized by the sgRNA (or crRNA), limiting its potential for directed evolution . Therefore, several editing tools were invented for the specific DNA segment mutagenesis (Table ).…”

Section: Constructions Of Be-based Microbial Gene-editing Toolsmentioning

confidence: 99%

“…BEs allow edition at the target sites which were hybridized by the sgRNA (or crRNA), limiting its potential for directed evolution. 70 Therefore, several editing tools were invented for the specific DNA segment mutagenesis (Table 2). A mutagenesis tool EvolvR, which employed an nCas9 domain fused to an error-prone DNA polymerase (Figure 3E), was able to induce mutations within the DNA sequence downstream of replicators.…”

Section: Base Editors Based On Cytosine-deamination Activity Of Tadamentioning

confidence: 99%

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

Qin

Zhang

et al. 2023

ACS Synth. Biol.

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Base editors (BE) based on CRISPR systems are practical gene-editing tools which continue to drive frontier advances of life sciences. BEs are able to efficiently induce point mutations at target sites without double-stranded DNA cleavage. Hence, they are widely employed in the fields of microbial genome engineering. As applications of BEs continue to expand, the demands for base-editing efficiency, fidelity, and versatility are also on the rise. In recent years, a series of optimization strategies for BEs have been developed. By engineering the core components of BEs or adopting different assembly methods, the performance of BEs has been well optimized. Moreover, series of newly established BEs have significantly expanded the base-editing toolsets. In this Review, we will summarize the current efforts for BE optimization, introduce several novel BEs with versatility, and look forward to the broadened applications for industrial microorganisms.

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Genome editor-directed in vivo library diversification

Cited by 9 publications

References 55 publications

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

Engineering, applications, and future perspectives of GPCR‐based genetically encoded fluorescent indicators for neuromodulators

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

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