Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

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Science  05 Aug 2016:
Vol. 353, Issue 6299, aad5147
DOI: 10.1126/science.aad5147

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Structured Abstract


Prokaryotes have evolved multiple systems to combat invaders such as viruses and plasmids. Examples of such defense systems include receptor masking, restriction-modification (R-M) systems, DNA interference (Argonaute), bacteriophage exclusion (BREX or PGL), and abortive infection, all of which act in an innate, nonspecific manner. In addition, prokaryotes have evolved adaptive, heritable immune systems: clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated proteins (CRISPR-Cas). Adaptive immunity is conferred by the integration of DNA sequences from an invading element into the CRISPR array (adaptation), which is transcribed into long pre-CRISPR RNAs (pre-crRNAs) and processed into short crRNAs (expression), which guide Cas proteins to specifically degrade the cognate DNA on subsequent exposures (interference).


A plethora of distinct CRISPR-Cas systems are represented in genomes of most archaea and almost half of the bacteria. The latest CRISPR-Cas classification scheme delineates two classes that are each subdivided into three types. Integration of biochemistry and molecular genetics has contributed substantially to revealing many of the unique features of the variant CRISPR-Cas types. Additionally, structural analysis and single-molecule studies have further advanced our understanding of the molecular basis of CRISPR-Cas functionality. Recent progress includes relevant steps in the adaptation stage, when fragments of foreign DNA are processed and incorporated as new spacers into the CRISPR array. In addition, three novel CRISPR-Cas types (IV, V, and VI) have been identified, and in particular, the type V interference complexes have been experimentally characterized. Moreover, the ability to easily program sequence-specific DNA targeting and cleavage by CRISPR-Cas components, as demonstrated for Cas9 and Cpf1, allows for the application of CRISPR-Cas components as highly effective tools for genetic engineering and gene regulation in a wide range of eukaryotes and prokaryotes. The pressing issue of off-target cleavage by the Cas9 nuclease is being actively addressed using structure-guided engineering.


Although our understanding of the CRISPR-Cas system has increased tremendously over the past few years, much remains to be revealed. The continuing discovery of CRISPR-Cas variants will provide direct tests of the recently proposed modular scenario for the evolution of CRISPR-Cas systems. The recent discovery and characterization of new CRISPR-Cas types with previously unknown features implies that our current knowledge has relatively limited power for predicting the functional details of distantly related CRISPR-Cas variants. Hence, newly discovered CRISPR-Cas systems need to be dissected thoroughly to gain insight into their biological roles, to unravel their molecular mechanisms, and to harness their potential for biotechnology. Key outstanding questions regarding CRISPR-Cas biology include the ecological roles of microbial adaptive immunity, the high rates of CRISPR-Cas horizontal transfer, and the coevolution of CRISPR-Cas and phage-encoded anti-CRISPR proteins. Relatively little is known about the regulation of CRISPR-Cas expression, and about the roles of CRISPR-Cas in processes other than defense. With respect to the CRISPR-Cas mechanism, details illuminating the connection between the adaptation stage and the interference stage in primed spacer acquisition remain elusive. A key aspect of CRISPR-Cas that is poorly understood at present is self/nonself discrimination. The discrimination mechanisms seem to differ substantially among CRISPR variants. Recent comparison of class 2 type effector complexes (Cas9/Cpf1) has revealed overall architectural similarities as well as structural and mechanistic differences, as had previously been found for the distinct types of class 1 effector complexes (Cascade/Cmr). These variations may translate into complementary biotechnological applications. As well as innovative tools for basic research, CRISPR-associated effector complexes will be instrumental for developing the next generation of antiviral prophylactics and therapeutics. For applications in human gene therapy, improved methods for efficient and safe delivery of Cas9/Cpf1 and their guide RNAs to cells and tissues are still needed. Further insight into the basic details of CRISPR-Cas structure, functions, and biology—and characterization of new Cas effector proteins in particular—is crucial for optimizing and further expanding the diverse applications of CRISPR-Cas systems.

Evolution of CRISPR-Cas systems resulted in incredible structural and functional diversity.

Class 1 CRISPR-Cas systems are considered to be the evolutionary ancestral systems. The class 2 systems have evolved from class 1 systems via the insertion of transposable elements encoding various nucleases, and are now being used as tools for genome editing.


Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications.

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