Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

2020/03/26 by Russell T. Walton, Kathleen A. Christie, Madelynn N. Whittaker +1 · 23 citations
Agricultural and Biological Sciences · Biochemistry, Genetics and Molecular Biology · #CRISPR and Genetic Engineering #Insect symbiosis and bacterial influences #RNA and protein synthesis mechanisms

paper · doi:10.1126/science.aba8853

openalex publication_date 2020/03/26 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/28

Abstract

Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

Citations

• A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

Cited by

• Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana
• Easy‐Curing and pH ‐Regulated CRISPR ‐Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris
• CRISPR base editor screening identifies spectrum of MEN1 mutations impacting menin inhibitors in clinical trials
• And… cut! – how conformational regulation of CRISPR-Cas effectors directs nuclease activity
• CRISPR/Cas-Based Epigenome Editing: Advances, Applications, and Clinical Utility
• Efficient in situ epitope tagging of rice genes by nuclease-mediated prime editing
• Engineering Herbicide Cross‐Resistance in Rapeseed by Generating Stacked BnaALS Mutations via Sequential CBE and ABE8e ‐ SpRY Editing
• Treating genetic blood disorders in the era of CRISPR-mediated genome editing
• Turbo‐charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond
• Towards precision medicine for brain arteriovenous malformations
• Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a
• Systematic discovery of CRISPR-boosted CAR T cell immunotherapies
• In vivo adenine base editing ameliorates Dravet syndrome phenotypes in a mouse model
• Base editing rescues seizures and sudden death in a SCN8A mutation-associated developmental epileptic encephalopathy model
• Expanding the flexibility of base editing for high-throughput genetic screens in bacteria
• Prime editing: Emerging mechanisms, engineering innovations, and next-generation applications
• Sequence-Independent RNA Sensing in Living Mammalian Cells
• Flexibility in PAM recognition expands DNA targeting in xCas9
• Precise generation of bystander-free mouse models with ABE9-SpRY
• Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi
• Template-independent genome editing and restoration for correcting frameshift disorders
• Therapeutic adenine base editor with minimized off-target effects
• PepMLM: Target Sequence-Conditioned Generation of Therapeutic Peptide Binders via Span Masked Language Modeling

Related

• Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering
• Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects
• Flexibility in PAM recognition expands DNA targeting in xCas9
• Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a
• Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a