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Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems

Author:
Gramelspacher, Max J., Hou, Zhonggang, Zhang, Yan
Source:
Methods 2019
ISSN:
1046-2023
Subject:
Archaea, CRISPR-Cas systems, DNA, DNA damage, adaptive immunity, bacteria, enzyme activity, eukaryotic cells, gene editing, genome, interspersed repetitive sequences, messenger RNA, prokaryotic cells, ribonucleases
Abstract:
The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9′s RNA-targeting potential.
Agid:
6484597