Question

2.  As you have seen, different Cas proteins from different sources are being used in CRISPR-based applications.  

a) Briefly compare and contrast Cas9, Cas12, and Cas13.

b) What feature of Cas12 and Cas13, absent from Cas9, makes these proteins particularly suitable for biosensing applications?

c) In what ways was Cas9 modified to turn it into a base editor?

Answer 1

Cas 9- The first and best-characterized single-protein CRISPR effector is Cas9. It makes a blunt double-stranded DNA break, which can then be repaired by either non-homologous end joining or homologous recombination with a donor template DNA to create site-specific edits. Type II-A Cas9s generally have high genome editing efficiency, but off-target cleavage at unintended genome sites can be a disadvantage.

Cas 12- Cas12 is a compact and efficient enzyme that creates staggered cuts in dsDNA. Cas12 processes its own guide RNAs, leading to increased multiplexing ability. Cas12 has also been engineered as a platform for epigenome editing, and it was recently discovered that Cas12a can indiscriminately cut single-stranded DNA once activated by a target DNA molecule matching its spacer sequence. This property makes Cas12a a powerful tool for detecting tiny amounts of target DNA in a mixture.

Cas 13- Cas13 is an outlier in the CRISPR world because it targets RNA, not DNA. Once it is activated by a ssRNA sequence bearing complementarity to its crRNA spacer, it unleashes a nonspecific RNase activity and destroys all nearby RNA regardless of their sequence. This property has been harnessed in vitro for precision diagnostics. These systems can also be used for efficient, multiplexable, and specific RNA knockdown or RNA sequence editing in mammalian cells. This makes Cas13 a potentially significant therapeutic for influencing gene expression without altering genome sequence.

b. The main requirements for biosensor applications are the identification of a target molecule, availability of a suitable biological recognition element, and the potential for disposable portable detection systems to be preferred to sensitive laboratory-based. Both Cas 12 and Cas 13 has RNAse activity which is missing in case 9 and probably this can be used for targeting certain areas.

c. Base editing is a newer genome editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks.

In RNA guided catalytically deficient Cas9 nuclease fused with a deaminase, specifically deaminates the target base. The modified base is read by DNA polymerases on the template DNA strand and the desired base pair change is introduced in the genome.