ABSTRACT
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein system is an adaptive immune system in prokaryotic organisms. It defends against invading bacteriophages and conjugative plasmids. In 2013, multiple research groups repurposed the CRISPR-Cas system for sequence-specific genome editing and transcriptional regulation in both prokaryotes and eukaryotes. Extensive studies on Cas9 protein engineering, gRNA modification, optimization of gRNA design and screening tools, have evolved the dominant Cas9 genome editing tool to be a reliable, versatile, and robust system with an exceptional editing efficiency and accuracy. Immunization and immunity are two steps of the CRISPR-Cas mechanism by which prokaryotes become resistant to foreign nucleic acids. Compared to these mainstream nucleotide-directed targeted genetic engineering tools for genome editing and gene regulation in bacteria, the major advantage of CRISPR-Cas system is the simplicity of the design and the potential of application in multiplexed editing and genome-scale mutagenesis.
