Type III CRISPR Systems

Introduction

Transimission Electron Micrograph of Bacteriophage inserting genome into E.coli. Source EYE OF SCIENCE/SCIENCE PHOTO LIBRARY

By Amir Brivanlou, 21'


Clustered Regularly Spaced Palindromic Repeats (CRISPR) are DNA sequences found throughout prokaryotes and archaea that, in conjunction with a variety of Cas enzymes, are responsible for anti-phage defense mechanisms. Generally, foreign bacteriophage DNA is recognized by the enzymes Cas1 and Cas2 and incorporated into the CRISPR array within the host organisms’ genome. These portions of bacteriophage DNA are known as spacers. These spacers can then be transcribed into RNA and used as guides to direct the cleavage of future phage invaders .

There exists a tremendous diversity of CRISPR systems, there are two distinct classes of CRISPR systems each with three types of CRISPR/Cas systems containing their own specialized Cas enzymes and modes of action . The type II CRISPR system, encoding a Cas9 endonuclease, is the best characterized of the CRISPR systems due to its prevalence as a gene-editing tool. The discovery and subsequent characterization of type II CRISPR systems and their use in gene editing recently resulted in the Nobel prize being awarded to Jennifer Doudna and Emmanuelle Charpentier .

While most CRISPR systems target DNA, type III CRISPR-Cas immunity has been shown to target both DNA and RNA, making them of special interest . Additionally, type III systems are thought to be the most ancient of the CRISPR systems as Cas10, the signature endonuclease of type III systems, was likely the original effector in bacteria and archaea . Type III CRISPR systems are thought to be the most complex of the CRISPR types, and in the following article I hope to highlight the current knowledge on spacer acquisition, biogenesis, and interference in type III CRISPR systems.

Class I and Class II CRISPR systems

There exist two distinct classes of CRISPR systems in bacteria and archaea. The first class contains types I, III, and IV, while the second includes types II, V, and VI. The key differences between these classes lie in the organization of the effector module. Type I systems possess a multi-protein crRNA-binding complex that functions together to target and cleave phage DNA. This heterogeneous effector complex in class I systems is functionally analogous to the Cas9, Cas12, or Cas13 effector modules in Class 2 systems which bind crRNAs, target phage DNA, and cleave it.

All CRISPR/Cas systems, however, must possess proteins that can execute the following four functions. The first fundamental function is “spacer adaptation,” or the process by which DNA sequences derived from cleaved prophage are incorporated into the CRISPR array. These genes usually encode an integrase, while Type III systems also often include an RT and integrase. The second function all CRISPR systems must possess is “expression,” which refers to the processing of the guide crRNAs, which are transcribed sequences of prophage DNA used to target future phage invaders. These crRNAs require extensive processing before they can be loaded into a nuclease and direct cleavage. Cas6 is the primary crRNA processor in Class I systems, while RNAse III or the endonuclease itself processes crRNAs in Class II systems. The third function of CRISPR systems is interference. Interference is the directed cleavage of prophage DNA. As previously stated, interference is executed by a single endonuclease in Class II systems, while several enzymes mediate class I system interference. Finally, the fourth function is loosely defined as “signal transduction,” these are helper proteins that are loosely linked to CRISPR immunity which are involved in metabolic regulation and general bacterial function. Despite common fundamental functions, there is tremendous diversity in how different CRISPR/Cas types guard the cell against phage. This article will focus on how Type III CRISPR systems execute these fundamental functions in unique ways.