CRISPR genome editing using a combined positive and negative selection system

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas is a key genome editing tool.
• Single Nucleotide Polymorphisms (SNPs) represent common human genetic variations, but many have unknown significance.
• Current CRISPR technology has limitations in precise single nucleotide substitutions.

Purpose of the Study:

• To develop an improved method for generating single base edits in the genome.
• To overcome limitations of existing CRISPR-based single nucleotide editing techniques.
• To investigate the biological relevance of variants of unknown significance.

Main Methods:

• Development of a Multiple Expression and Dual Selection (MEDS) system.
• Integration of MEDS with CRISPR-Cas9 technology.
• Utilized cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) for negative selection and neomycin phosphotransferase II (NPT II) for positive selection.

Main Results:

• Demonstrated the feasibility of the MEDS system for generating single base edits.
• Successfully introduced a specific point mutation (A→T) in the hemoglobin beta gene.
• Generated sickle hemoglobin as a proof of concept for the MEDS protocol.

Conclusions:

• The MEDS system enhances the feasibility of generating precise single nucleotide edits.
• This approach offers a valuable alternative for genome editing, complementing CRISPR/Cas.
• The method holds potential for studying genetic variants of unknown significance and their roles in disease.