2. As you have seen, differen
2. As you have seen, different Cas proteins fromdifferent sources are being used in CRISPR-basedapplications.
a) Briefly compare and contrast Cas9, Cas12, and Cas13.
b) What feature of Cas12 and Cas13,absent from Cas9, makes these proteins particularly suitable forbiosensing applications?
c) In what ways was Cas9 modified toturn it into a base editor?
Answer:
Cas 9- The first and best-characterized single-protein CRISPReffector is Cas9. It makes a blunt double-stranded DNA break, whichcan then be repaired by either non-homologous end joining orhomologous recombination with a donor template DNA to createsite-specific edits. Type II-A Cas9s generally have high genomeediting efficiency, but off-target cleavage at unintended genomesites can be a disadvantage.
Cas 12- Cas12 is a compact and efficient enzyme that createsstaggered cuts in dsDNA. Cas12 processes its own guide RNAs,leading to increased multiplexing ability. Cas12 has also beenengineered as a platform for epigenome editing, and it was recentlydiscovered that Cas12a can indiscriminately cut single-stranded DNAonce activated by a target DNA molecule matching its spacersequence. This property makes Cas12a a powerful tool for detectingtiny amounts of target DNA in a mixture.
Cas 13- Cas13 is an outlier in the CRISPR world because ittargets RNA, not DNA. Once it is activated by a ssRNA sequencebearing complementarity to its crRNA spacer, it unleashes anonspecific RNase activity and destroys all nearby RNA regardlessof their sequence. This property has been harnessed in vitro forprecision diagnostics. These systems can also be used forefficient, multiplexable, and specific RNA knockdown or RNAsequence editing in mammalian cells. This makes Cas13 a potentiallysignificant therapeutic for influencing gene expression withoutaltering genome sequence.
b. The main requirements for biosensor applications are theidentification of a target molecule, availability of a suitablebiological recognition element, and the potential for disposableportable detection systems to be preferred to sensitivelaboratory-based. Both Cas 12 and Cas 13 has RNAse activity whichis missing in case 9 and probably this can be used for targetingcertain areas.
c. Base editing is a newer genome editing approach that usescomponents from CRISPR systems together with other enzymes todirectly install point mutations into cellular DNA or RNA withoutmaking double-stranded DNA breaks.
In RNA guided catalytically deficient Cas9 nuclease fused with adeaminase, specifically deaminates the target base. The modifiedbase is read by DNA polymerases on the template DNA strand and thedesired base pair change is introduced in the genome.
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