A programmable pAgo nuclease with high activity and specificity for efficient DNA and RNA manipulation

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Prokaryotic Argonaute proteins (pAgos) are programmable nucleases with potential for nucleic acid manipulation.
• Existing pAgos often show target specificity for DNA or RNA and limited efficacy on high GC-content dsDNA.
• Development of versatile nucleases is crucial for advancing biotechnology tools.

Purpose of the Study:

• To characterize GgeAgo, a pAgo from the thermophilic Geobacillus genomosp. 3.
• To evaluate GgeAgo's DNA and RNA cleavage capabilities, including its performance on dsDNA with varying GC content.
• To assess GgeAgo's utility in practical biotechnological applications.

Main Methods:

• Characterization of GgeAgo activity using 5' phosphorylated DNA guides.
• Testing cleavage efficiency on DNA and RNA targets under diverse conditions.
• Assessing performance on plasmid and linear dsDNA with varying GC content, including at elevated temperatures.
• Evaluating GgeAgo in applications like DNA cloning, nucleic acid detection, and variant enrichment.

Main Results:

• GgeAgo efficiently cleaves both DNA and RNA with high specificity.
• GgeAgo effectively cleaves dsDNA with GC content up to 53% at elevated temperatures, retaining activity at 64% GC.
• GgeAgo demonstrated robust performance in DNA cloning, nucleic acid detection, and rare variant enrichment with high sensitivity and specificity.

Conclusions:

• GgeAgo is a versatile prokaryotic Argonaute protein with broad DNA and RNA cleavage activity.
• GgeAgo overcomes limitations of previous pAgos regarding GC content and temperature stability.
• GgeAgo shows significant potential as a valuable tool for diverse DNA and RNA manipulation tasks in biotechnology.