PiRun Zhang ¹ Wenli Zhang ^2* Jiahuan Li ^3* Huiying Liu ^4* Yantong Yu ^5* Xiaoping Yang ^4* Wenqing Jiang ^4*

• ¹ Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China
• ² Qingdao Mental Health Center, Qingdao, Shandong Province, China
• ³ Qingdao Central Hospital, Qingdao, Shandong Province, China
• ⁴ Qingdao Haici Hospital, Qingdao, China
• ⁵ Qingdao Traditional Chinese Medicine Hospital, Qingdao, Shandong Province, China

Rampant C-to-U RNA editing drives the mutation and evolution of SARS-CoV-2. While much attention has been paid to missense mutations, the C-to-U events leading to AUG and thus creating novel ORFs were uninvestigated. By utilizing the public time-course mutation data from world-wide SARS-CoV-2 population, we systematically identified the "AUG-gain mutations" caused by C-to-U RNA editing. Synonymous mutations were of special focus. Totally 58 synonymous C-to-U sites are able to create out-of-frame AUG in CDS. These 58 synonymous sites showed significantly higher allele frequency (AF) and increasing rate (dAF/dt) than other C-to-U synonymous sites in SARS-CoV-2 population, suggesting that these 58 AUG-gain events conferred additional benefits to the virus and are subjected to positive selection. The 58 predicted new ORFs created by AUG-gain events showed the following advantages compared to random expectation: they have longer lengths, higher CAI, higher Kozak scores, and higher tAI. The 58 putatively novel ORFs have high expressibility and are very likely to be functional, providing an explanation for the positive selection on the 58 AUG-gain mutations. Our study proposed a possible mechanism of the emergence of de novo genes in SARS-CoV-2. This idea should be helpful in studying the mutation and evolution of SARS-CoV-2.