Table 1.

Pros and cons of each circRNA preparation method for practical use.

Method	Pros	Cons
Chemical-based ligation	• Various chemical reactions	• Toxicity concerns
		• Uses DNA splint
	• No intronic scar	• Multiple steps • Unnatural bond
Enzyme-based ligation	• No intronic scar (however, the first 2~3 nucleotides are guanosine by T7, SP6, T3 RNA polymerase)	• Uses DNA or RNA splint • Ligases from T4 bacteriophage [52] require 5′-monophosphate
	• Large GOI	• Multiple steps • Ligation efficiency
Ribozyme-based ligation	• Relatively simple • Large GOI	• Intronic scar by group I intron PIE • Larger transcript including ribozyme
	• Reactions in vitro & in vivo	• RNA contaminants after reaction