Set 2: CRISPR Mechanisms and Classifcation

Briefly, what are the 3 stages of CRISPR-Cas immunity?

Adaptation - Cas1-Cas2 capture viral DNA and insert new spacer into CRISPR array via DNA repair.

Expression - CRISPR locus is transcribed and processed from preRNA to crRNAs/

Interference - crRNA guides an effector complex to viral DNA which is then cleaved.

Describe and compare the effector complexes in class 1 and 2 CRISPR systems?

Effector modules are highly diverse (unlike adaptor modules cas1 and cas2), their variation forms classification of CRISPR systems. All systems are divided into class 1 or 2. Class 1 have multi-subunit effector complexes comprised of several Cas proteins. Class 2 has a single, large multi domain effector protein.

Describe and compare the pre-crRNA processing in class 1 and 2 CRISPR systems?

Class 1 and 2 systems differ in the mechanisms of pre-crRNA processing.

Class 1 - crRNAs are generated by a dedicated complex of multiple Cas proteins.

Class 2 - processing is catalysed by either an external bacterial enzyme RNase III with the help of an additional RNA species (the transacting CRISPR tracr RNA) or by the same effector protein involved in target cleavage.

Describe and compare the interference stages in class 1 and 2 CRISPR systems?

During interference stage, in class 1, the processing complex containing the guide crRNA recognises the target site and recruits an additional Cas protein (cas3 in type I, Cas 10 in type II) that contains the nuclease domain directly responsible for target cleavage. In class 2 cleavage is performed by nuclease domain of large effector protein.

What are the major similarities and differences in class 1 compared to class 2?

Class 1 have a multi subunit effector complex, use Cas 3 or Cas 10 and have a separate nuclease which is recruited.

In contrast, class 2 has a single multi domain effector protein, use either Cas9, Cas12 or Cas13 and the effector contains a nuclease domain.

They differ in effector architecture, crRNA processing and interference mechanisms. Similarities include they both use Cas1 and Cas2 during adaptation.

Why does CRISPR qualify as ‘adaptive immunity’ system in prokaryotes?

It remembers prior exposure by acquiring a spacer from an invader, inserts into chromosome, and passes on to daughter cells, generating a sequence-specific and heritable protective response.

Before 2007, adaptive immunity was thought to be unique to vertebrates.

How does CRISPR adaptation work, and what is the role of Cas1-Cas2?

Adaptation is where new spacers are added from invading DNA.

The Cas1-Cas2 complex captures a short fragment of DNA (~22nt) and inserts it at the leader-proximal end of the CRISPR array, duplicating the adjacent repeat (~30nt). If Cas1 or Cas2 are deleted, no spacers are acquired and immunity is lost. The new spacer becomes part of the chromosome and is inherited by daughter cells.

Describe the structure of the Cas1-Cas2 complex and describe the DNA substrate?

Cas1 forms dimers, in which one of the two active sites bind DNA. Cas2 doems a dimer creating a spacer of correct length. The final complex is (Cas1)4-(Cas2)2.

Substrate DNA is ~23 bp dsDNA with short single strand flaed ends for easier access. It is called pre-spacer and proto-spacer. Cas1 active sites bind DNA ends with PAM specificity.

What is the mechanism for spacer capture in adaptation?

1. viral DNA enters the cell

2. Cas1-Cas2 binds to fragment of DNA

3. dsDNA ends are splayed by tyrosine wedges in each Cas1 dimer which lock open the DNA branch points while fixing in place a core 23-base pair dsDNA region

4. 3' ssDNA extends into active subunits of each Cas1 dimer for integration.

5. Length of spacer is governed by the fixed distance between 2 Cas1 wedges and from the branch points to integration sites - 'molecular ruler'.

6. Integration at leader-repeat junction

Briefly describe spacer integration?

Integration occurs at leader-junction on a chromosome, as a result a new spacer is added and a new repeat is duplicated. PCR evidence shows a ~60bp increase per new spacer (including repeats). This is inherited by daughter cells.

Typically, how long are spacers and repeats?

spacers are ~22 bp long, repeats are ~30 bp long.