A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction

TY - JOUR

T1 - A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction

AU - Han, Wenyuan

AU - Li, Yingjun

AU - Deng, Ling

AU - Feng, Mingxia

AU - Peng, Wenfang

AU - Hallstrøm, Søren

AU - Zhang, Jing

AU - Peng, Nan

AU - Liang, Yun Xiang

AU - White, Malcolm F.

AU - She, Qunxin

PY - 2017/2/28

Y1 - 2017/2/28

N2 - The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of IIIB Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5α-tag of cr-RNA and the 3α-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.

AB - The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of IIIB Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5α-tag of cr-RNA and the 3α-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.

U2 - 10.1093/nar/gkw1274

DO - 10.1093/nar/gkw1274

M3 - Journal article

C2 - 27986854

SN - 0305-1048

VL - 45

SP - 1983

EP - 1993

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 4

M1 - gkw1274

ER -