Stable gene targeting in human cells using single-strand oligonucleotides with modified bases

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Multiplexed genome engineering in E. coli uses oligonucleotides for simultaneous multi-locus editing.
• Similar technology in human cells could accelerate functional genomics and variant-phenotype studies.
• Current oligo-mediated human cell targeting faces low efficiency and poor cell survival.

Purpose of the Study:

• To enhance oligonucleotide-mediated genome engineering efficiency and cell survival in human cells.
• To investigate methods for improving oligo-targeting by addressing mismatch repair and toxicity.
• To establish a foundation for rapid and scalable genome engineering in human cells.

Main Methods:

• Utilized a HeLa-based enhanced green fluorescent protein (EGFP)-rescue reporter system.
• Employed modified base analogs in oligonucleotides to bypass mismatch repair.
• Investigated oligonucleotide toxicity by correlating phosphorothioate bond number with survival rates.
• Conducted comparative RNA-sequencing (RNA-seq) analysis to assess cellular responses.

Main Results:

• Modified base analogs increased oligo-mediated targeting efficiency.
• Reduced phosphorothioate bonds in oligonucleotides decreased toxicity.
• Achieved stable EGFP-corrected cells at a frequency of approximately 0.05% with optimized oligonucleotides.
• RNA-seq data suggested that cellular immunity contributes to low viability of oligo-corrected cells.

Conclusions:

• Optimized oligonucleotide design combining modified bases and reduced phosphorothioate bonds significantly improves genome engineering in human cells.
• Addressing oligonucleotide toxicity and potential immune responses is crucial for enhancing cell survival.
• This optimized method holds promise for rapid and scalable genome engineering applications in human cells, advancing functional genomics research.