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The Biology of CRISPR-Cas: Backward and Forward Assignment Victoria Matthews CRISPR-Cas defense utilizes crRNAs for sequence specific targeting of invading genetic elements. Precursor crRNA usually lies within the leader sequence after the CRISPR array. This transcript generates mature crRNAs that are composed of a repeat segment that is recognized by Cas proteins in a structure. Type 1 and 2 systems both employ Cas6 enzymes in order to specifically process the repeat within the pre-crRNA. The majority of type 1 precrRNAs have palindromic sequences within their repeats. They are then able to perform stable stem-loop structures that are recognized by Cas6 or Cas5d. These nucleases cut the RNA downstream of the hairpin. This yields mature crRNAs that are composed of a full spacer flanked by a short repeat-derived 5’ handle and 3’ stem loop. The cas6 enzymes stay bound to the crRNA after cleavage and act as scaffolds for the creation of a cascade. Some type 2 repeat sequences can be unstructured and rely on Cas6 to remodel crRNA. The type 2 Cas6 proteins show high sequence similarity to Cas6 homologs of type 1, but type 2 Cas6 proteins are not part of the interference complex. Class 2 systems take over the interference machinery and non-Cas proteins for crRNA maturation. Type V-B and type 2 systems need tracrRNA for CRISPR mediated immunity. Cas9 of type 2A stabilizes the tracrRNA crRNA duplex, which recruits the host protein RNase 3 for processing. RNase then removes the 5’ repeat-derived tag, now the duplex is ready for interference. Type 3 systems utilize the tracrRNA crRNA duplex for target interference. The repeats of the type 2C arrays contain promoter elements that transcribe individual crRNAs. These crRNAs can be further processed by RNase 3. Cas12 and Cas13 have dual nuclease activity for target interference and crRNA processing with type V and type IV systems. Cas12a recognizes the repeat hairpin structure and cleaves the repeat the create crRNAs with 5’ repeat-derived tags with type VA systems. Type VI processing does not require tracrRNA for crRNA processing. This is because Cas13a recognizes the structure of the repeat within the pre-crRNA and process it upstream of the hairpin. Cas13b mediated processing results in the creation of mature crRNAs that harbor a 3-nucleotide spacer portion. This has been shown to promote target cleavage by the effector....