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Analysis of conventional and alternative CRISPR/Cas9 genome editing to enhance a single-base pair knock-in mutation

Authors
  • Edmondson, Carina1
  • Zhou, Qi1
  • Liu, Xuan1
  • 1 University of California, Riverside, CA, 92521, USA , Riverside (United States)
Type
Published Article
Journal
BMC Biotechnology
Publisher
Springer (Biomed Central Ltd.)
Publication Date
Jul 27, 2021
Volume
21
Issue
1
Identifiers
DOI: 10.1186/s12896-021-00707-5
Source
Springer Nature
Keywords
Disciplines
  • Research
License
Green

Abstract

BackgroundThe use of CRISPR/Cas9 technologies in generating single-base pair knock-in mutations has recently exploded in the number of methods available. However, with the growing expansion of new technologies, it can be difficult to determine the best method for genome editing.ResultsIn this study, we evaluated a number of CRISPR/Cas9 approaches for deriving cell lines with knock-in base pair edits to create a phosphorylation mutation and provide a breakdown of editing efficiencies and suggestions for improvement. Overall, our studies suggest that using pre-formed ribonucleoprotein (RNP) complexes is a reliable editing method to generate homozygous single-base pair mutations. We also show that antibiotic selection coupled homologous recombination is an efficient tool for generating highly specific heterozygous mutations.ConclusionThe methods and/or combination of methods outlined in this study can be used to help other researchers with similar goals in single-base pair genome editing.

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