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

Abstract

Background

The 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.

Results

In 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.

Conclusion

The 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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