Addressing the dNTP bottleneck restricting prime editing activity.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Cellular Dynamics

Background:

• Prime editing, a versatile gene editing tool, exhibits limited efficiency in quiescent or slowly proliferating cells due to strict regulation of intracellular deoxynucleotide triphosphate (dNTP) levels.
• The Moloney murine leukemia virus (MMLV)-reverse transcriptase, a key component of prime editors, necessitates high dNTP concentrations for optimal DNA polymerization and editing activity.

Approach:

• Investigated mutations designed to enhance the enzymatic properties of the MMLV-reverse transcriptase subunit of prime editors.
• Explored the complementary strategy of targeting SAMHD1 (a dNTPase) for degradation to increase intracellular dNTP pools.
• Evaluated the combined impact of these approaches on prime editing efficiency in primary cells and in vivo.

Key Points:

• Specific mutations were introduced into the MMLV-reverse transcriptase to improve its catalytic efficiency and dNTP utilization.
• Degradation of SAMHD1 was shown to effectively increase intracellular dNTP levels, a critical factor for prime editor function.
• The combination of enhanced MMLV-reverse transcriptase and SAMHD1 degradation significantly boosted prime editing outcomes.

Conclusions:

• Engineered prime editors with improved MMLV-reverse transcriptase enzymatic properties demonstrate enhanced activity in challenging cellular contexts.
• Targeting SAMHD1 for degradation represents a viable strategy to overcome dNTP limitations and further augment prime editing efficiency.
• These advancements hold promise for improving the in vivo and primary cell applicability of prime editing for therapeutic and research purposes.