Upon entering the cell, the invader DNA is recognized and degraded. A piece of the invader DNA (shown in red) which is adjacent to a PAM sequence (shown in light blue) is selected and integrated into the CRISPR locus as a new spacer. The CRISPR locus consists of the leader sequence (L), which contains the promoter (black arrow), short repeat sequences (shown in black) and spacer sequences (shown in light grey). The new spacer (shown in red) is integrated at the 5′-end of the CRISPR locus.
(A) The cas gene cluster is located on the mini-chromosome pHV4 and codes for the proteins Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7 and Cas8b, with the last-named being the signature protein for subtype I-B. The cluster is flanked by two CRISPR loci (P1 and P2). CRISPR locus P1 contains 17 repeats and 16 spacers (shown in dark grey and light grey respectively), and locus P2 contains 12 repeats and 11 spacers. The third CRISPR locus is encoded on the main chromosome and contains 25 repeats and 24 spacers. The leader region (L) at the 5′-end of the CRISPR locus contains the promoter (black arrow). (B) The repeat sequences of the three different crRNAs differ by one residue. The sequence of the CRISPR locus C repeat is shown. Positions that are identical in locus P1 and P2 repeats are displayed with a dot.
Figure 3(A) The invader plasmid and (B) mutations that impair the CRISPR/Cas defence
(A) To challenge the defence system of Haloferax, invader plasmids were constructed which carry the first spacer of the CRISPR locus P1 and trinucleotide sequences adjacent to the spacer as potential PAM sequences. Initially, trinucleotide sequences were cloned on both sides of the spacer until our results showed that the PAM sequence is located upstream. PyrE2-deficient Haloferax cells were transformed with the 62 different invader plasmids and plated on selection medium. Only cells that retain the plasmid can able to grow on the selection medium. A valid PAM triggers the defence reaction, which then results in degradation of the plasmid and a drastically reduced transformation efficiency (at least 100-fold). (B) H. volcanii colonies that grew up on selection medium in spite of being transformed with an invader plasmid having a functional PAM were analysed for mutations in the CRISPR/Cas genes. For each clone, the cas gene cluster and spacer sequences in the CRISPR locus as well as on the invader plasmid were all sequenced. Most of the 30 clones analysed had deletions in the cas genes (57%), and mutations in the cas genes were also observed (20%). A few had the spacer sequence in the CRISPR locus P1 deleted (10%), and some had lost the spacer sequence on the invader plasmid (7%). One had the functional PAM sequence mutated (3%) rendering it non-functional, and for one mutant (3%) we did not find any changes, either in the cas gene cluster, or in the spacer sequences in the CRISPR locus, or on the invader plasmid.