It is at this moment that chaperone activity could be required to allow a smooth exit from the ER and prevent a blockade induced by exposed hydrophobic domains. Base pairing with the DNA insertion site in the first place and then catalyzing insertion and reverse transcription of the intron RNA by RNP confer specificity upon the subsequent integration event. To achieve rapid and efficient selection of positive integrants, a retro transposition activated selectable Atractylenolide-III marker was introduced into intron domain IV. However, this strategy cannot be used to isolate clones containing a second intron insertion in an already erythromycin-resistant mutant. To solve this problem, RAM was flanked by two repeated FLP recognition target sites and it can be removed from the chromosome in FLP recombinase-mediated step. Therefore, the authors proposed that this system could be applied for insertional mutation of multiple gene in Clostridium. Nevertheless, this approach would leave an intron residual fragment of over 0.9 kb in the genome. The disruption of the other genes by this system in an already intron insertion mutant might lead to the instability of the previously mutated genes due to the presence of LtrA, through which the excision of a DNA sequence flanked by two intron fragments might occur via homologous recombination. Nevertheless, follow-up researchers suggested that this method is of low reproducibility and laborious to screen for double-crossover integration events. Moreover, this strategy would leave an erythromycin resistance marker in the genome for screening, which prevents it from manipulating multiple genes, since not many markers are available for Clostridia. The aim of this study was to develop a more efficient targeted gene deletion strategy for Clostridium. By combining the principles of the ����ClosTron���� system and homologous recombination, we developed an accurate gene deletion procedure which enabled us efficiently delete DNA fragments without leaving any antibiotic resistance marker in the genome. This strategy might aid in the genetic dissection of clostridial virulence and engineering industrial clostridial strains for efficient production of biofuels and biobased chemicals. Targeted gene deletion via double crossover recombination remains a challenge for Clostridium. Diatrizoic acid ClosTron has been adopted for targeted gene disruption in Clostridium, with an integration frequency of nearly 100% in some clostridial species.