CRISPR–Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9, this protein/gRNA complex degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were reported, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. Recent data indicated that expression of AcrIIA4 with Cas9 could modulate Cas9/gRNA complex activities for controlling the outcomes of Cas9-mediated gene editing.

Full-length AcrIIA4 cDNA (86 aa) was constructed with codon optimization using gene synthesis technology and expressed with a small T7-His-TEV cleavage site Tag (29aa) fusion at its N-terminal and 11 Poly-Arginine (11R) tag at its C-terminal. This protein was expressed in E. coli as inclusion bodies. The final product was refolded using our unique “temperature shift inclusion body refolding” technology and chromatographically purified.

May be used for in vitro modulating Cas9 protein activities study for gene editing experiments by intracellular delivery of this AcrIIA4-11R protein directly adding protein into cell culture medium.