Part:BBa_K2996001

cas1-cas2 complex

Mechanism

Cas1-Cas2 is a heterohexamer containing a Cas2 dimer sandwiched by two Cas1 dimers, where Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. When double-stranded breaks (DSBs) form, which is much more common in foregin DNA RecBCD binds the free DNA ends and processes them into prespacers.

The optimal substrate for E. coli Cas1-Cas2 is a 33-bp prespacer, which contains a central 23-bp double-stranded duplex with 5-nt overhangs on each side splayed into single-stranded DNA ends by tyrosine wedges in Cas1.The length of the prespacer is determined by the distance between two Cas1 integrase active sites. The 23-bp duplex sits on the flat surface of Cas1-Cas2, which positions the 3′ends into the catalytic site of each Cas1. In the E. coli type I-E system, Cas1-Cas2 complex which has high binding affinity for prespacer substrates with a canonical PAM (5′-CTT-3′).

Once loaded with the prespacer,Cas1-Cas2 recognizesthe CRISPR array and catalyzes spacer integration. 3 ′-OH group of the prespacer attacks the backbone at the leader-proximal end of the first repeat with in the CRISPR array,forming a half-site intermediate.The end of the prespacer containing the cytosine from the PAM attacks at the leader-distal end of the opposite strand of the repeat, forming a full-site intermediate.The single-stranded repeats on either end of the integrated spacer are filled by DNA polymeraseI,and the nicks are ligated to create an intact CRISPR array.

In the end, the CRISPR will have an addition of 61 base pairs.

Usage

The constructed pRec plasmid can express Cas1 and Cas2 upon addition of anhydrotetracycline (aTc), which results in spacer acquisition, as the information input. Similar to pLux-cas plasmid, addition of AHL molecules will lead to the expression of Cas1-Cas2 complex.