ArticleScientific reports2021
Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum.
Article in Scientific reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 19 citations in OpenAlex.
- Advances in prime editing: Molecular innovations, Large-fragment engineering, and AI-driven design.Biodesign research · 2026Review
- Expanding the Microbial Genomic Landscape and Biotechnological Applications of CRISPR-Cas Systems.Biology · 2026Review
- Genome editing across Dictyostelia species enables comparative functional genetics of social amoebas.Scientific reports · 2026Article
- Long Non-Coding RNAs in the Cold-Stress Response of Horticultural Plants: Molecular Mechanisms and Potential Applications.International journal of molecular sciences · 2025Review
- Optogenetic control of cAMP oscillations reveals frequency-selective transcription factor dynamics in Dictyostelium.Development (Cambridge, England) · 2025Article
- Efficient endogenous protein labelling in Dictyostelium using CRISPR/Cas9 knock-in and split fluorescent proteins.PloS one · 2025Article
- Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling.Stem cell reviews and reports · 2024Article
- Genome editing.Scientific reports · 2022Article
- CRISPR/Cas9-based genome-wide screening of Dictyostelium.Scientific reports · 2022Article
- Can SpRY recognize any PAM in human cells?Journal of Zhejiang University. Science. B · 2022Article
- Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques.Frontiers in plant science · 2022Review
- Moving the Research Forward: The Best of British Biology Using the Tractable Model SystemCells · 2021Review
- Review
- CRISPR Toolbox for Genome Editing inFrontiers in cell and developmental biology · 2021Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.
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