In this study, the CRISPR/Cas3 system from Pseudomonas aeruginosa type I-C is presented as a new tool for genome editing applicable in various bacteria. Compared to the “classic” Cas9 gene scissors from Streptococcus pyogenes, Cas3 has two distinct functions: on the one hand, Cas3 cuts a single strand of DNA in its function as a nuclease (cutting component) and, on the other hand, Cas3 also has a function as a helicase. Helicases unravel the DNA double helix. With this function, Cas3 creates the basis for the progressive deletion of large DNA regions. Specific „signposts“ called crRNAs are used to find the target region(s) to be deleted in the genome. A signpost is integrated into a so-called Cascade complex (consists of Cas5, Cas8 and Cas7), which functions as the recognition component of the CRISPR/Cas3 system. Once the Cascade complex with the crRNA has found the appropriate target region in the genome, Cas3 is recruited and, starting from the target area, begins to unwind and cut the DNA in both directions. This results in deletions of large areas of DNA. One of the authors of the study describes the CRISPR/Cas3 system as a kind of “Pac Man” who chews up the DNA .
The scientists used the CRISPR/Cas3 system in P. aeruginosa to induce deletions in different areas of the bacterial genome. They showed that, starting from the respective target region, large deletions that differ in size can be generated very efficiently. The efficiency of CRISPR/Cas3 can be impaired by essential (vital) genes that lie in the vicinity of the target area. If these genes are deleted by CRISPR/Cas3, these bacteria either die or grow more slowly.
A direct comparison with the CRISPR/Cas9 system showed that the activity of CRISPR/Cas3 mainly results in large deletions. CRISPR/Cas9, on the other hand, produces predominantly short deletions and point mutations or the bacterial genome remains unedited. It was also shown that using CRISPR/Cas3 and the addition of a DNA template, that matched neighboring areas of the target region, can be used to induce targeted deletions.
The CRISPR/Cas3 system is also used in three other bacteria (Escherichia coli, Pseudomonas syringae, and Klebsiella pneumoniae) to create large deletions in their genomes. The plant pathogen P. syringae does not have a CRISPR/Cas system naturally. In the genome of P. syringae there are many genes that convey the virulence, i.e. the infectious power, of the pathogen. These genes are organized redundantly in so-called gene clusters and are difficult to delete with previous techniques. Using the CRISPR/Cas3 system, large regions of the genome were deleted that contained the virulence clusters. The scientists also used a crRNA to delete two different gene clusters at the same time (a so-called multiplexed targeting approach).
The CRISPR/Cas3 system is the naturally most common CRISPR/Cas system in bacteria. Thus, lots of bacteria have their own CRISPR/Cas3 system built into their genome. The scientists therefore also used bacterial strains that have their own CRISPR/Cas3 system and inserted only crRNAs for target regions. The result: large deletions were also produced using bacteria with an endogenous CRISPR/Cas3 system.
According to the publication, the CRISPR/Cas3 system described here can be used in synthetic biology and, for example, to reduce the size of the genome of bacteria and to remove large regions of the genome. In that way, bacteria can be synthesized in which pathogenic and/or redundant DNA regions have been deleted. The authors also envision an application in higher organisms (plants, animals, humans), for example to delete areas of the genome in which there are hardly any genes and of which it is not yet known why they are present in the genome. Furthermore, CRISPR/Cas3 could also be used to delete highly repetitive DNA sequences.
So far it is not fully understood how the CRISPR/Cas3 reaction in the genome is terminated. Different bacteria have different DNA repair mechanisms, that can be involved in that process. In P. aeruginosa, small repeating DNA sequences can often be found at the ends (so-called microhomologies) or there are no clearly matching DNA sequences. The observed microhomologies suggest that MMEJ (microhomology-mediated end joining) repair is involved in these bacteria. In P. syringae and E. coli, larger homologous DNA sequences can be found at the ends, which indicates that HDR (homology directed repair) is involved.
Another naturally occurring system could interfere with the expectations of the scientists: So-called anti-CRISPR proteins, which are found in bacteriophages, can inactivate CRISPR/Cas3 in bacteria, that are infected by bacteriophages. The scientists introduced a gene into the bacteria whose gene product is supposed to block these anti-CRISPR proteins. With this “anti-anti-CRISPR” they were able to circumvent this problem.
The study impressively demonstrates how different CRISPR/Cas systems and their modes-of-action are in bacteria and how these systems can be used for genome editing. Open questions regarding possible unintended changes in the genome as induced by CRISPR/Cas3 activity and the repair processes involved remain open.