Genomic and Transcriptomic Analyses of Prime Editing Guide RNA-Independent Off-Target Effects by Prime Editors

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• Prime editors (PEs) enable targeted genomic modifications.
• RNA-guided genome editors can cause guide-dependent off-target mutations.
• The potential for guide-independent off-target effects from PE reverse transcriptase activity in mammalian cells remains unexamined.

Purpose of the Study:

• To systematically investigate guide-independent off-target mutations in prime editor 3 (PE3)-edited human cells.
• To assess potential DNA/RNA mutations, telomere alterations, retroelement activity, alternative splicing, and gene expression changes.
• To evaluate the specificity of the PE reverse transcriptase moiety.

Main Methods:

• Development of a cost-effective, sensitive platform for profiling guide-independent off-target effects.
• Analysis of DNA and RNA in prime editor 3 (PE3)-edited human cells.
• Assessment of telomeres, endogenous retroelements, alternative splicing, and gene expression.

Main Results:

• No guide-independent off-target mutations were detected in the DNA or RNA of PE3-edited cells.
• No significant alterations were observed in telomeres, endogenous retroelements, alternative splicing, or gene expression.
• The study demonstrated undetectable guide RNA-independent off-target effects.

Conclusions:

• Prime editor 3 (PE3) exhibits high specificity in human cells, with no detectable guide RNA-independent off-target effects.
• The reverse transcriptase moiety of PE3 does not appear to introduce guide-independent mutations.
• These findings support the safety and specificity of prime editing technology.