Anti-CRISPR type II subtype A4; AcrIIA4 is an anti-CRISPR (Acr) protein which was discovered via a self-targeting method that inhibits the type II-A CRISPR-Cas system (Csy). AcrIIA4 interacts with Cas9-single-guide RNA (sgRNA) and inhibits Cas9 activity by structurally mimicking the protospacer adjacent motif (PAM) to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by Cas9. AcrIIA4 further inhibits the endonuclease activity of Cas9 by shielding its RuvC active site. The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Co-expression of Cas9 and AcrIIA4 which is fused with the N terminal region of human Cdt1 (AcrIIA4-Cdt1) not only increases the frequency of homology-directed repair (HDR) but also suppresses off-targets effects. As AcrIIA4 interferes with Cas9 DNA binding activity, it may have use as a CRISPR controller of Cas9-mediated genome engineering: chemically inducible anti-CRISPR protein AcrIIA4 has been engineered to disable Cas9 DNA binding upon the addition of trimethoprim. Cas9-based application of interest to the field of population control is that of the 'gene drive'; CRISPR-Cas9 gene drives have been developed towards reducing the burden of pests and vector-borne diseases such as human malaria vector Anopheles gambiae. Germline expression of a phage-derived AcrIIA4 inactivated CRISPR-based gene drives and restore their inheritance to Mendelian rates in A. gambiae; and AcrIIA4 and AcrIIA2 proteins inhibit active gene drive systems in budding yeast. Almost all type II Acr proteins characterized to date directly interact with the Cas9 endonuclease, although by distinct mechanisms. The type II CRISPR/Cas subtype has a distinct crRNA-guided surveillance complex encoded by cas9 (formerly csn1), cas1, cas2, and csn2 (for type IIA) or cas4 (for type IIB) genes, type II-Cs are generally characterized by the absence of both cas4 and csn2; these genes are located all located in a single transcriptional unit directly upstream of the CRISPR locus. Cleavage of the DNA target in type II systems is carried out by Cas9 which is an RNA-guided double-stranded DNase with two independent nuclease domains, HNH and RuvC. Due to the reliance of the type II system on a single protein for function, Cas9 homologs derived from different subtypes and species have been utilized for numerous gene editing applications. CRISPR-Cas immune systems are used by certain prokaryotes and archaea to resist the invasion of foreign nucleic acids such as phages or plasmids. Anti-CRISPRs are small proteins which are the natural inhibitors for CRISPR-Cas systems; encoded on bacterial and archaeal viruses, they allow the virus to evade host CRISPR-Cas systems. The CRISPR-Cas-mediated adaptive immune response can be divided into three steps, including the acquisition of spacer derived from invading nucleic acids, crRNA processing, and target degradation. Theoretically, Acr proteins could suppress any step to disrupt the CRISPR-Cas system. Acr proteins are diverse with no common sequence or structural motif, and they inhibit a wide range of CRISPR-Cas systems with various inhibition mechanisms. CRISPR-Cas systems are divided into two classes (1 and 2) and six types (class 1: types I, III and IV; class 2: types II, V and VI). Class 2 systems employ a single multi-domain effector Cas9 protein complex that performs target recognition and cleavage. Acr families are named for their type and subtype which are numbered sequentially as they are discovered.

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                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*.
gi 1220112056  8 REIKNKDYTVKLSGTDS-NSITQLIIRVNNDGNEYVISESENESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITL 82
Cdd:cd22281    1 REIKNLEYEIKAKGYDStNSITQLVINVNGDGNEYPISESEYESIVEKFIETFKNGWNGTYEDEEEFYNDMQEIAL 76

Anti-CRISPR type II subtype A4; AcrIIA4 is an anti-CRISPR (Acr) protein which was discovered via a self-targeting method that inhibits the type II-A CRISPR-Cas system (Csy). AcrIIA4 interacts with Cas9-single-guide RNA (sgRNA) and inhibits Cas9 activity by structurally mimicking the protospacer adjacent motif (PAM) to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by Cas9. AcrIIA4 further inhibits the endonuclease activity of Cas9 by shielding its RuvC active site. The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Co-expression of Cas9 and AcrIIA4 which is fused with the N terminal region of human Cdt1 (AcrIIA4-Cdt1) not only increases the frequency of homology-directed repair (HDR) but also suppresses off-targets effects. As AcrIIA4 interferes with Cas9 DNA binding activity, it may have use as a CRISPR controller of Cas9-mediated genome engineering: chemically inducible anti-CRISPR protein AcrIIA4 has been engineered to disable Cas9 DNA binding upon the addition of trimethoprim. Cas9-based application of interest to the field of population control is that of the 'gene drive'; CRISPR-Cas9 gene drives have been developed towards reducing the burden of pests and vector-borne diseases such as human malaria vector Anopheles gambiae. Germline expression of a phage-derived AcrIIA4 inactivated CRISPR-based gene drives and restore their inheritance to Mendelian rates in A. gambiae; and AcrIIA4 and AcrIIA2 proteins inhibit active gene drive systems in budding yeast. Almost all type II Acr proteins characterized to date directly interact with the Cas9 endonuclease, although by distinct mechanisms. The type II CRISPR/Cas subtype has a distinct crRNA-guided surveillance complex encoded by cas9 (formerly csn1), cas1, cas2, and csn2 (for type IIA) or cas4 (for type IIB) genes, type II-Cs are generally characterized by the absence of both cas4 and csn2; these genes are located all located in a single transcriptional unit directly upstream of the CRISPR locus. Cleavage of the DNA target in type II systems is carried out by Cas9 which is an RNA-guided double-stranded DNase with two independent nuclease domains, HNH and RuvC. Due to the reliance of the type II system on a single protein for function, Cas9 homologs derived from different subtypes and species have been utilized for numerous gene editing applications. CRISPR-Cas immune systems are used by certain prokaryotes and archaea to resist the invasion of foreign nucleic acids such as phages or plasmids. Anti-CRISPRs are small proteins which are the natural inhibitors for CRISPR-Cas systems; encoded on bacterial and archaeal viruses, they allow the virus to evade host CRISPR-Cas systems. The CRISPR-Cas-mediated adaptive immune response can be divided into three steps, including the acquisition of spacer derived from invading nucleic acids, crRNA processing, and target degradation. Theoretically, Acr proteins could suppress any step to disrupt the CRISPR-Cas system. Acr proteins are diverse with no common sequence or structural motif, and they inhibit a wide range of CRISPR-Cas systems with various inhibition mechanisms. CRISPR-Cas systems are divided into two classes (1 and 2) and six types (class 1: types I, III and IV; class 2: types II, V and VI). Class 2 systems employ a single multi-domain effector Cas9 protein complex that performs target recognition and cleavage. Acr families are named for their type and subtype which are numbered sequentially as they are discovered.

                         10        20        30        40        50        60        70
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*.
gi 1220112056  8 REIKNKDYTVKLSGTDS-NSITQLIIRVNNDGNEYVISESENESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITL 82
Cdd:cd22281    1 REIKNLEYEIKAKGYDStNSITQLVINVNGDGNEYPISESEYESIVEKFIETFKNGWNGTYEDEEEFYNDMQEIAL 76