ArticleNature communications2021
Engineered dual selection for directed evolution of SpCas9 PAM specificity.
Article in Nature communications, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 25 citations in OpenAlex.
- PAM-flexible SpCas9 variants expand the targeting scope for porcine genome editing and cellular disease modeling.BMC biotechnology · 2026Article
- Advances in Double-Stranded DNA Targeting Technologies.Exploration (Beijing, China) · 2026Review
- Directed evolution expands CRISPR-Cas12a genome-editing capacity.Nucleic acids research · 2025Article
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- Article
- CRISPR/Cas9: a sustainable technology to enhance climate resilience in major Staple Crops.Frontiers in genome editing · 2025Review
- SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping.Nature communications · 2024Article
- Engineered transcription-associated Cas9 targeting in eukaryotic cells.Nature communications · 2024Article
- Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells.Nature communications · 2024Article
- Engineered transcription-associated Cas9 targeting in eukaryotic cells.bioRxiv : the preprint server for biology · 2024Article
- Base Editors-Mediated Gene Therapy in Hematopoietic Stem Cells for Hematologic Diseases.Stem cell reviews and reports · 2024Review
- Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri.Mikrochimica acta · 2024Article
- Engineering Cas9: next generation of genomic editors.Applied microbiology and biotechnology · 2024Review
- DNA double-strand break-free CRISPR interference delays Huntington's disease progression in mice.Communications biology · 2023Article
- Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor.Nature communications · 2021Article
- Evolution-aided engineering of plant specialized metabolism.aBIOTECH · 2021Review
Corrections and comments
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Authors and funding
8 authors at 1 institution in 1 country.
Funding
Abstract
The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.
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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.