731 Type VI A Cas13a In Cas13a direct repeat region of crRNA is composed of 5 6

7.3.1Type VI-A (Cas13a)In Cas13a, direct repeat region of crRNA is composed of 56 bp stem using Watsonand Crick base pairing with an unpaired 2 nt bulge (AA or AC) present at 30end ofstem, while loop region of stem contains 79 nt depending upon homologs ofCas13a. In addition, an 812 nt single-stranded region is present at 50-end of thestem-loop structure. This single-stranded region is important for the recognition ofcrRNA by Cas13a (Liu et al.2017a) (Fig.7.4).7.3.2Type VI-B (Cas13b)Although detailed structure of crRNA for type VI-B does not exist, predictivemodels of secondary structure showed that compared with other types (VI-A,VI-B, and VI-C), stem structure of DR in VI-B is longer (914 bp), with bulgesand a smaller loop structure. In addition, in contrast to other types, in which DRrepeat is present at 50end of the crRNA-spacer, VI-B contains DR region at 30-end ofthe crRNA-spacer (OConnell2019) (Fig.7.4).7.3.3Type VI-C (Cas13c) and Type VI-D (Cas13d)Compared with type VI-A and VI-B, DR region in type VI-C is much smaller inlength comprising 30 nt (Figure). The stem-loop structure in type VI-C crRNAcomprises a stem of 9 bp with 45 nt loop. In addition, a 3 nt shortanking sequence4-7 basal bp. Stem3-6 nt. Loop9-14 total stem bp.14-16 nt. total stem lengthProcessing SiteType VI-B3’5’Processing Site5’ FlankLoopcrRNA-SpacerLba-crRNA Directed-Repeat (DR)Type VI5’3’Stem3’- Bulge7-9 nt. Loop20-30 nt.3’-Spacer*5-6 bp. Stem1-5 nt. mature5’ flank5’Processing Sites2 nt. 3’bulge3’Type VI-AType VI-C20-30 nt.3’-Spacer*9 bp.Stem#4-5 nt. Loop3 nt.3’ Flank3 nt.5’ Flank5’3’8-10 bp.Stem #5 nt.3’ FlankProcessing SiteType VI-D20-30 nt.3’-Spacer*4-5 nt. Loop1-5 nt. mature5’ flank5’3’2 nt.3’-Bulge20-30 nt.5’-Spacer*Key:=Processing site is different from otherorthologs of Cas13*=Spacer length can vary due to trimmingby host nuclease#= Stem contain bulges or mismatchesFig. 7.4Different subtypes of type VI system: the important similarities and differences betweencrRNAs from each of the different type VI system subtypes232S. Ashraf et al.

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