Classification of CRISPR-CAS System

Classification of CRISPR-CAS System

Ever since its discovery the CRISPR CAS System has become a vital tool in the field of life sciences. What was originally discovered as bacterial immunity system in the bacteria is now being used in the field of life sciences as a broad range genome editing tool. After scientist successfully hijacked this immunity system and turned it in to a genome editing tool, it has opened a new horizon in the field of biological sciences but it was a tough journey. One of the basic challenges faced by the scientists was to characterize various types of CRISPR Systems, their characterization was a big challenge thanks to extensive exchange of CAS genes and gene modules among various bacteria.

Scientists across the globe performed number of genomic analysis in order to establish a classification system capable of broadly identifying various types of CRISPR CAS systems. After that position specific scoring matrices (PSSM) of all the known CAS systems were developed. Then only those CAS locus were considered for further classification which were declared complete. A CRISPR-CAS system was declared as complete if it posses the adaptation modules and complete set of genes required for the formation of interference module. Keeping in view all these findings CRISPR-CAS system was divided on the basis of what type of interference module is encoded by its genes. Those encoding for a multi subunit CrRNA complex were placed in “Class-1” CRISPR systems and those encoding for a single multipurpose interfering module were placed in “Class-2” CRISPR-CAS systems. Each class has further types and subtypes.

The Class-1 has:  

•  Type I
•  Type III
•  Type IV

The Class-2 has:  

• Type II
• Type V

Class 1:

The class 1 systems are described as those types of systems which require a large complex of multiple proteins to conduct the interference procedure. The class 1 has further 3 types The Type I, Type III and Type IV systems.

   v  Type I:

          The type I system is further divided in to a total of 7 subtypes namely sub-type, I-A, I-B, I-C, I-D, I-E, I-F, and I-U. All kinds of type I locus consists of a gene that encodes for the signature protein CAS 3 or its variant the CAS 3’. This furthers promotes the formation of helicase  which causes unwinding of double stranded DNA or the DNA-RNA complex. The helicase is fused with endonucleases which causes the cleavage of target DNA. Many of the subtypes are typically encoded by a single operon which encodes for cas1, cas2, cas3 and genes for cascade complex subunits.

  v  Type III:

             All the type III CAS systems are characterized by the presence of a specific protein called the CAS 10 protein which is the signature for the Type III systems. This CAS 10 induces formation of a multi-domain protein containing palm domain which is the largest subunit of the crRNA-effector complex. The type III systems are also reported to produce a CAS7 and a CAS5 protein subunit. This complex is further fused with specific nucleases which cuts and denatures the enzyme.

  v  Type IV:

              The type IV CAS system is one of the two new CRISPR CAS systems discovered in the recent studies. In this type the “csf1” protein serves as the signature protein. The specific function of this system type is still uncharacterized but how it functions? That has been sorted out recently; it has a multi-subunit crRNA-effector consisting of Csf1, Cas5 and Cas7 protein subunit. In addition to that it also contains a Ding family helicase or a alpha-helicase.

Class 2:

These type of systems are describes as those type of CRISPR-CAS system in which the entire procedure of interference is carried out by a single large proteinic molecule.  The class 2 has 2 types.

  v  Type II:

            Type II system is the most widely studied system because of the famous CAS9 belonging to this type. The signature gene for this system is the CAS9 which encodes for a single multi-domain protein which carries out the interference function. The system also contains CAS1 and CAS2 genes which carries out the adoption stage and is often assisted by the CAS9 protein. The type II system is further subdivided in to 3 subtypes; subtype II-A, II-B, and II-C. The II-A subtype posses an additional “csn2” gene, it has been reported to assist in spacer acquisition. The subtype II-B lacks csn2 but it has a “cas4” gene. The subtype II-C has only three genes the CAS1, CAS2 nad CAS9.

3D model of CAS9

  v  Type V:

            Type V is one of the two newly discovered CRISPR systems (type IV being the other newly discovered system). This system is characterized by having a special gene called the “cpf1” gene, which encodes for a large protein of about 1300 amino acids. This system type was first discovered in prevotella and francesella bacterial species. In similarity to CRISPR CAS9 all the interference process is conducted by a single large protein module named the CPF1. However the cpf1 lacks a n HNH nucleases domain which is common in CAS9 protein. Also the cpf1 is encoded outside the CRISPR-CAS context in several genomes which indicates it to be a possible addition because of transposable elements. In addition to cpf1 it also encodes for a cas1, cas2 and in rare cases cas4 proteins. Unlike other Class II systems which are specific to bacteria the cpf1 has been reported in one archae as well.

 

3D model of CPF1

Conclusion:

                         Till now scientists have successfully classified various CRISPR-CAS system in to 2 classes, 5 types and 16 subtypes, on the basis of their genetic characters and protein modules produced by the genes. This classification is vital for scientists as it will enable them to further enhance their knowledge, easily identifying and placing new CRISPR systems and implementing newly discovered systems for further high tech research.