Epitranscriptomic reprogramming in response to low CO₂ stress and m⁶A engineering to enhance biomass production in...

N⁶-adenine methylation (m⁶A) as an epitranscriptomic mark is the most abundant modification in eukaryotic RNA and plays a dynamically regulated role. However, m⁶A dynamics, deposition and engineering in microalgae remain largely unknown. Here, in Nannochloropsis oceanica, the dynamic alterations and reprogramming in m⁶A RNA modifications after the shift from high to low CO₂ conditions were first investigated using methylated RNA immunoprecipitation sequencing. The m⁶A peaks in N. oceanica were mainly enriched in 3'UTR. A positive association between m⁶A abundance and mRNA transcription of CO₂-responsive genes was observed; moreover, N. oceanica cells adopted versatile strategies in a dynamic reprogramming of m⁶A in response to low CO₂ stress. Secondly, knockout of two putative m⁶A methylases including NoMTA (NO04G02990) and NoMTB (NO07G02450) by genome editing induced methylation reprogramming, which was associated with expression changes of low-CO₂ responsive genes such as carbon/nitrogen metabolism, and photorespiration genes that underlie reductions in growth and biomass. Lastly, m⁶A modification reprogramming was first engineered to increase low-CO₂ stress tolerance and biomass productivity by the CRISPR/dCas13 system combined with MTA and NoMTB under low CO₂ in N. oceanica. Therefore, these strides would pave the way for microalgal epigenetics and future industrial applications.