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Protocol for assessment of CRISPR base editors and their components in Escherichia coliopen access
- Authors
- Shelake, Rahul Mahadev; Kim, Jae-Yean
- Issue Date
- Sep-2025
- Publisher
- Cell Press
- Keywords
- Biotechnology And Bioengineering; Crispr; Gene Expression; Molecular Biology; Graphpad Prism Software; Crispr-cas System Guide Rna; Rifampicin Derivative; Article; Bacterial Strain; Clinical Assessment; Clustered Regularly Interspaced Short Palindromic Repeat; Computer Model; Controlled Study; Crispr Cas System; Cytotoxicity Assay; Dna Binding; Dna Extraction; Dna Fragmentation; Dna Isolation; Dna Sequencing; Dna Transcription; Dna Transfer; Electroporation; Enzyme Activity; Escherichia Coli; Expression Vector; Fluorescence Activated Cell Sorting; Fluorescence Analysis; Gene Editing; Gene Expression; Gene Location; Gene Mutation; Gene Sequence; Genetic Transcription; Homologous Recombination; Nonhuman; Plasmid; Polyacrylamide Gel Electrophoresis; Polymerase Chain Reaction; Protein Expression; Saccharomyces Cerevisiae; Sanger Sequencing
- Citation
- STAR Protocols, v.6, no.3
- Indexed
- SCOPUS
ESCI
- Journal Title
- STAR Protocols
- Volume
- 6
- Number
- 3
- ISSN
- 2666-1667
2666-1667
- Abstract
- Base editing (BE) is a CRISPR technique that allows precise nucleobase conversions. However, high expression of BE components is often toxic in Escherichia coli. Here, we present a protocol for analyzing BE at single or multiple target sites using promoter-terminators for single guide RNA (sgRNA) and BE component expression. We describe steps for designing and cloning sgRNA, synthetic target, and BE biomodules. We then detail procedures for BE module assemblage, E. coli transformation, and testing base editors and components. For complete details on the use and execution of this protocol, please refer to Shelake and Kim,1 Shelake et al.,2 and Shelake et al.3
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