ArticleNature biotechnology2016
Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.
Article in Nature biotechnology, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 122 papers.
What it found
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Who cites it
122 citing papers in PubMed.
- Strategies and mechanisms of precision genome engineering: From gene editing to genome writing.iMetaOmics · 2026Review
- Azide-functionalized SpCas9 enables generation of site-selective and bioactive Cas9-siRNA conjugates.Chemical communications (Cambridge, England) · 2026Article
- Using Domain Insertion to Create Sulfite Reductases That Present Chemical-Dependent Activities.ACS synthetic biology · 2026Article
- High-throughput engineering of ligand-activated splicing ribozyme through domain insertion.bioRxiv : the preprint server for biology · 2026Article
- A versatile method for designing biosensors via regulatory domains of allosteric enzymes.Nature communications · 2026Article
- Stage- and tissue-specific gene editing using 4-OHT-inducible Cas9 in whole organism.The Journal of cell biology · 2026Article
- QBEmax is a sequence-permuted and internally protected base editor.Nature biotechnology · 2026Article
- Structural and functional insights into internal domain replacement in SpCas9 for protein engineering.Scientific reports · 2025Article
- RESTRICT-seq enables time-gated CRISPR screens and uncovers novel epigenetic dependencies of SCC resistance.bioRxiv : the preprint server for biology · 2025Article
- Rational engineering of allosteric protein switches by in silico prediction of domain insertion sites.Nature methods · 2025Article
- Improving adenine base editing precision by enlarging the recognition domain of CRISPR-Cas9.Nature communications · 2025Article
- Autonomous Nucleic Acid and Protein Nanocomputing Agents Engineered to Operate in Living Cells.ACS nano · 2025Review
- Direct delivery of Cas-embedded cytosine base editors as ribonucleoprotein complexes for efficient and accurate editing of clinically relevant targets.Nucleic acids research · 2025Article
- Gene Editing: An Effective Tool for the Future Treatment of Kidney Disease.Journal of inflammation research · 2025Review
- Engineered transcription-associated Cas9 targeting in eukaryotic cells.Nature communications · 2024Article
- A compact, versatile drug-induced splicing switch system with minimal background expression.Cell reports methods · 2024Article
- Engineered transcription-associated Cas9 targeting in eukaryotic cells.bioRxiv : the preprint server for biology · 2024Article
- The art of designed coiled-coils for the regulation of mammalian cells.Cell chemical biology · 2024Review
- Rational Design of a Circularly Permuted Flavin-Based Fluorescent Protein.Chembiochem : a European journal of chemical biology · 2024Article
- Engineering self-deliverable ribonucleoproteins for genome editing in the brain.Nature communications · 2024Article
62 more citing papers are in PubMed but not listed here.
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Authors and funding
7 authors.
Funding
Abstract
The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.