Source 1primary paper2019Communications Biology
Toolkit/directed evolution
directed evolution
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Directed evolution is an engineering method that improves biological tool performance by iteratively selecting functional protein variants. In the cited split fluorescent protein study, it was demonstrated as one of two approaches used to improve split fluorescent proteins, contributing to brighter split sfCherry3 variants.
Usefulness & Problems
Why this is useful
Directed evolution is useful for enhancing protein function when rational prediction alone is insufficient. In the provided evidence, it enabled improvement of split fluorescent proteins that support endogenous protein tagging by gene editing and multiplexed visualization applications in living Caenorhabditis elegans.
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for multiplexed visualization of neuronal synapses in living C. elegans
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facilitating the tagging of endogenous proteins by gene editing
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Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
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CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes.
Problem solved
This method addresses the problem of suboptimal performance in engineered protein tools by enabling selection of improved variants from sequence diversity. In the cited context, it helped solve limited split fluorescent protein performance by improving split FP function and brightness.
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for multiplexed visualization of neuronal synapses in living C. elegans
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facilitating the tagging of endogenous proteins by gene editing
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Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
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CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes.
Published Workflows
Objective: Develop multiplexable acoustic reporter genes for two-tone ultrasound imaging of gene expression and demonstrate their use for distinguishing cell populations and states in vitro and in vivo.
Why it works: The workflow is presented as combining rational protein design and directed evolution to create reporter variants with distinct acoustic pressure-response profiles, which then enables two-tone ultrasound imaging.
acoustic discrimination based on pressure-response profilesrational protein designdirected evolutionultrasound imaging
Stages
- 1.Reporter engineering(library_design)
To overcome the single-sound limitation of prior ARGs by creating two distinguishable reporter variants.
Selection: Develop new ARG variants with distinguishable acoustic pressure-response profiles.
- 2.In vitro utility demonstration(functional_characterization)
To show that the engineered reporters are useful for distinguishing biological populations before in vivo application.
Selection: Assess whether multiplexed ARGs can delineate bacterial cell species and cell states in vitro.
- 3.In vivo application(in_vivo_validation)
To demonstrate that multiplexed ARG imaging works in relevant in vivo settings including the mouse gastrointestinal tract and tumor-colonizing bacterial agents.
Selection: Apply multiplexed ARGs to image distinct bacterial subpopulations in living mice.
Objective: Engineer AAV capsids for more precise and potent in vivo delivery to brain, muscle, and retina while reducing required vector dose.
Why it works: The abstract states that these approaches increasingly converge by integrating structural hypotheses, in vivo selections, and multi-trait computational optimization, implying that combining mechanistic design with empirical selection and computation improves capsid performance across tissues.
receptor-targeted BBB transport via TfR1 or ALPLfine-tuning HSPG interactions to facilitate ILM traversalliver detargeting for muscle-directed deliverydirected evolutionrational designmachine learningin vivo selectionsmulti-trait computational optimizationintegration of structural hypotheses
Objective: Outline a hybrid synthetic ALife design methodology that could enable on-demand bioengineering of problem-solving artificial life by addressing deficiencies in directed evolution.
Why it works: The proposed methodology is expected to work by combining the adaptive evolutionary strengths of digital ALife in simulated environments with synthetic biology methods for realizing engineered living systems, while addressing deficiencies in directed evolution, fitness mapping, and fitness estimation.
directed evolutiongenetic diversity generationfitness landscape mappingfitness estimationdigital in silico evolutionary designsynthetic in vitro evolutionary design
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete method used to build, optimize, or evolve an engineered system.
Mechanisms
selection of functional protein variantsTarget processes
No target processes tagged yet.
Implementation Constraints
The cited implementation context is protein engineering of split fluorescent proteins, where directed evolution was used alongside SpyTag/SpyCatcher-assisted complementation as an improvement strategy. The evidence does not provide construct design details, host systems for selection, library generation methods, or screening assay parameters.
The supplied evidence does not specify the mutagenesis scheme, screening workflow, number of rounds, or quantitative gains attributable specifically to directed evolution alone. Independent replication and performance across multiple tool classes are not established by the provided sources.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Source 2review2018International Journal of Genomics
Ranked Claims
NLG-1 CLASP enables multiplexed visualization of neuronal synapses in living C. elegans.
for multiplexed visualization of neuronal synapses in living C. elegans
NLG-1 CLASP enables multiplexed visualization of neuronal synapses in living C. elegans.
for multiplexed visualization of neuronal synapses in living C. elegans
NLG-1 CLASP enables multiplexed visualization of neuronal synapses in living C. elegans.
for multiplexed visualization of neuronal synapses in living C. elegans
NLG-1 CLASP enables multiplexed visualization of neuronal synapses in living C. elegans.
for multiplexed visualization of neuronal synapses in living C. elegans
NLG-1 CLASP enables multiplexed visualization of neuronal synapses in living C. elegans.
for multiplexed visualization of neuronal synapses in living C. elegans
NLG-1 CLASP enables multiplexed visualization of neuronal synapses in living C. elegans.
for multiplexed visualization of neuronal synapses in living C. elegans
NLG-1 CLASP enables multiplexed visualization of neuronal synapses in living C. elegans.
for multiplexed visualization of neuronal synapses in living C. elegans
The split sfCherry3 variants facilitate tagging of endogenous proteins by gene editing.
facilitating the tagging of endogenous proteins by gene editing
The split sfCherry3 variants facilitate tagging of endogenous proteins by gene editing.
facilitating the tagging of endogenous proteins by gene editing
The split sfCherry3 variants facilitate tagging of endogenous proteins by gene editing.
facilitating the tagging of endogenous proteins by gene editing
The split sfCherry3 variants facilitate tagging of endogenous proteins by gene editing.
facilitating the tagging of endogenous proteins by gene editing
The split sfCherry3 variants facilitate tagging of endogenous proteins by gene editing.
facilitating the tagging of endogenous proteins by gene editing
The split sfCherry3 variants facilitate tagging of endogenous proteins by gene editing.
facilitating the tagging of endogenous proteins by gene editing
The split sfCherry3 variants facilitate tagging of endogenous proteins by gene editing.
facilitating the tagging of endogenous proteins by gene editing
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Based on sfCherry3, the authors developed a new red-colored trans-synaptic marker called NLG-1 CLASP.
Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
Based on sfCherry3, the authors developed a new red-colored trans-synaptic marker called NLG-1 CLASP.
Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
Based on sfCherry3, the authors developed a new red-colored trans-synaptic marker called NLG-1 CLASP.
Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
Based on sfCherry3, the authors developed a new red-colored trans-synaptic marker called NLG-1 CLASP.
Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
Based on sfCherry3, the authors developed a new red-colored trans-synaptic marker called NLG-1 CLASP.
Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
Based on sfCherry3, the authors developed a new red-colored trans-synaptic marker called NLG-1 CLASP.
Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
Based on sfCherry3, the authors developed a new red-colored trans-synaptic marker called NLG-1 CLASP.
Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP)
CRISPR-Cas9 has been used across applications including gene therapy, gene regulation, epigenome modification, and chromosome imaging.
CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes.
These Cas9 limitations hinder the use of CRISPR for disease treatment and wider biotechnological applications.
These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications.
Cas9 can exhibit aberrant off-target activity.
(ii) aberrant off-target activity
Cas9 has a strict dependence on a protospacer-adjacent motif sequence.
some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence
Cas9 lacks sufficient modulation of protein binding and endonuclease activity for precise spatiotemporal control.
(iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing
The large size of Cas9 creates problems for CRISPR delivery.
(iii) the large size of Cas9 is problematic for CRISPR delivery
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
Approval Evidence
2 sources4 linked approval claimsfirst-pass slug directed-evolution
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
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studies that improve or develop novel protein functions through ... directed evolution
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method improvementsupports
SpyTag/SpyCatcher interaction and directed evolution were demonstrated as two approaches to improve split fluorescent proteins.
we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution
Source:
performance improvementsupports
Directed evolution yielded two split sfCherry3 variants with substantially enhanced overall brightness.
The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Source:
review focussupports
The review emphasizes domain fusion or splitting, rational design, and directed evolution as protein-engineering strategies for expanding SpCas9 versatility.
Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
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strategy overviewsupports
Protein-engineering approaches are presented as solutions to overcome Cas9 limitations and generate more robust and efficient DNA manipulation tools.
Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation.
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Comparisons
Source-backed strengths
The evidence supports directed evolution as an experimentally demonstrated route for improving split fluorescent proteins. Its practical strength here is association with improved split sfCherry3 performance and downstream utility in endogenous protein tagging and synapse visualization.
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The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness
Ranked Citations
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