Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CRISPAIR (Study of the interplay between CRISPR interference and DNA repair pathways towards the development of novel CRISPR tools)

Teaser

Over the last few years CRISPR-Cas systems have emerged as a powerful tool to edit genomes, control gene expression and much more. These technologies have promising applications ranging from gene therapy to cure genetic disease to new therapies against cancer and infectious...

Summary

Over the last few years CRISPR-Cas systems have emerged as a powerful tool to edit genomes, control gene expression and much more. These technologies have promising applications ranging from gene therapy to cure genetic disease to new therapies against cancer and infectious diseases. CRISPR-Cas loci are originally the adaptive immune system of archaea and bacteria. They can capture pieces of invading DNA such as bacteriophages and use this information to degrade target nucleic acids through the action of RNA-guided nucleases. The consequences of DNA cleavage by Cas nucleases, i.e. how breaks are processed and whether they can be repaired, largely remains to be investigated. A better understanding of the interplay between DNA repair and CRISPR-Cas is critical both to shed light on the evolution and biology of these fascinating systems and for the development of biotechnological tools based on Cas nucleases. Genome editing strategies take advantage of different DNA repair pathways to introduce mutations upon DNA cleavage. In bacteria however, the introduction of breaks by Cas nucleases in the chromosome has been described to kill the cell. Using a combination of bioinformatics and genetics approaches we are investigating the interplay between CRISPR and DNA repair in bacteria with a particular focus on the widely used CRISPR-Cas9 system. We are also trying to understand what determines the differences in the activity of Cas9 at different target positions. The knowledge gained is helping us to develop novel tools for bacterial genome engineering, gene silencing, and should also contribute to the design of improved CRISPR antimicrobials able to efficiently eliminate virulent and antibiotic resistant bacteria specifically.

Work performed

Cas9 cleavage in the chromosome of bacteria has been reported to kill the cell. Bacteria mainly rely on homologous recombination with sister chromosomes to repair double strand breaks. We showed that the simultaneous cleavage of all copies of the Escherichia coli chromosome at the same position cannot be repaired, leading to cell death. Repair of double strand breaks can also be achieved through a pathway that does not rely on homologous recombination, known as NHEJ. In the pathway, the ends of the broken DNA can simply be stitched back together but in manner that frequently introduces mutations. The error-prone NHEJ repair pathway is conveniently used in Eukaryotic cells to introduce indels in target sequences and knockout genes. The ability of most eukaryotic cells to repair DNA breaks through NHEJ is presumably the reason why they are able to survive Cas9 breaks better than bacteria. NHEJ is indeed absent from many widely studied bacterial strains including E. coli. We wondered if the introduction of a heterologous NHEJ pathway in E. coli would be able to rescue cells from Cas9-mediated killing. The NHEJ pathway from Mycobacterium tuberculosis was successfully introduced in E. coli were it could repair Cas9 breaks but only with a very low efficiency.

We also wondered if the ability of NHEJ to repair Cas9 breaks could make CRISPR-Cas9 immunity against phages inefficient. We tested this idea by studying the patterns of co-occurrence of the two systems in bacterial genomes. We found that NHEJ and type II-A CRISPR-Cas systems only co-occur once among 5563 fully sequenced prokaryotic genomes. We then investigated experimentally the possible molecular interactions causing this negative association. Our results suggest that the NHEJ system has no effect on type II-A CRISPR-Cas interference and adaptation. On the other hand, we provide evidence for the inhibition of NHEJ repair by the Csn2 protein from type II-A CRISPR-Cas system. Our findings give insights on the complex interactions between CRISPR-Cas systems and repair mechanisms in bacteria and contribute to explain the scattered distribution of CRISPR-Cas systems in bacterial genomes.

The inactivation of a gene can either be achieved through the introduction of a mutation using genome editing techniques, or by directly blocking its expression. The catalytically dead variant of Cas9 known as dCas9 can indeed be guided by small RNAs to bind target DNA without cutting thereby blocking transcription in a strategy also known as CRISPRi. In a recent study we revealed that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We used this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrated broad applicability of this method to the study of genetic regulation and cellular physiology. We then used dCas9 to silence genes in high-throughput screens. High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. We used a genome-wide library of guide RNAs to direct dCas9 to block gene transcription in Escherichia coli. This screening method can help us understand how the cell works in a systematic way. Gene essential for bacterial growth can be identified and which could reveal interesting antibiotic targets. Finally, this study enabled to identify important design rules to use dCas9 in bacteria, including the existence of a surprising toxicity phenomenon when dCas9 is guided to target specific sequences.

Final results

The work performed so far has enabled to obtain a much better understanding of the properties of both Cas9 and dCas9 in bacteria. We have shown how the interplay between CRISPR-Cas9 systems and the DNA repair systems in bacteria not only determines the outcome of DNA cleavage events, with important applications to genome editing techniques, but also shapes the distribution of CRISPR-Cas systems across bacterial clades. We will now further investigate the interaction between other types of CRISPR-Cas systems and DNA repair pathways using both comparative genomics approaches and experimental validation.
We have also furthered our understanding of how dCas9 can block the RNA polymerase and can be used to precisely control gene expression. The application of dCas9 in high-throughput screens has revealed an unexpected toxicity phenomenon that we now aim to elucidate in the rest of the project. This will likely reveal interesting biology and let us develop more powerful screening strategies to study gene essentiality and genetic interaction networks.