ArticleNature methods2019
Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.
Article in Nature methods, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 34 papers.
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Who cites it
34 citing papers in PubMed.
- Chemical and Structural Engineering of Guide RNAs for Precision Genome Editing: From Design Principles to Clinical Applications.Chemical biology & drug design · 2026Review
- Article
- Decoding the role of chromatin context in the off-target effects of CRISPR gene editing with EGOLD.Cell discovery · 2026Article
- Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity.Nature communications · 2025Article
- A unified genetic perturbation language for human cellular programming.bioRxiv : the preprint server for biology · 2025Article
- CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction.Fundamental research · 2025Article
- A rational engineering strategy for structural dynamics modulation enables target specificity enhancement of the Cas9 nuclease.Nucleic acids research · 2025Article
- Engineering a New Generation of Gene Editors: Integrating Synthetic Biology and AI Innovations.ACS synthetic biology · 2025Review
- Resolving discrepancies between chimeric and multiplicative measures of higher-order epistasis.Nature communications · 2025Article
- Off-target sequence variations driven by the intrinsic properties of the Cas-sgRNA-DNA complex in genome editing.PloS one · 2025Article
- Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Mutational rescue of the activity of high-fidelity Cas9 enzymes.Cell reports methods · 2024Article
- Plant Functional Genomics Based on High-Throughput CRISPR Library Knockout Screening: A Perspective.Advanced genetics (Hoboken, N.J.) · 2024Article
- High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2.Nature biomedical engineering · 2024Article
- Engineering Cas9: next generation of genomic editors.Applied microbiology and biotechnology · 2024Review
- Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse".International journal of molecular sciences · 2023Review
- Quantification of evolved DNA-editing enzymes at scale with DEQSeq.Genome biology · 2023Article
- Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.Molecular therapy : the journal of the American Society of Gene Therapy · 2023Article
- A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets.Nature communications · 2023Article
- Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities.Nature communications · 2022Article
Corrections and comments
- Erratum issued
Authors and funding
12 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.
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Registered trials
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.