Periodic Reporting for period 4 - CRISPAIR (Study of the interplay between CRISPR interference and DNA repair pathways towards the development of novel CRISPR tools)
Periodo di rendicontazione: 2020-09-01 al 2022-08-31
Over the last few years CRISPR-Cas systems have been harnessed as a powerful tools to edit genomes, control gene expression, detect specific sequences and much more. These technologies have promising applications ranging from gene therapy to new treatments 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. The CRISPAIR ERC project enabled to obtain a better understanding of the consequences of Cas9 cleavage in the chromosome of bacteria and the interplay with various DNA repair pathways. The project further allowed us to establish the design rules for efficient targeting with Cas9, and in particular for efficient gene silencing through the use of the catalytically dead variant of Cas9 known as dCas9. This powerful method enables to silence any gene of interest in a bacterial genome to study its function, doing so with great precision and control. Finally, the knowledge gained during the CRISPAIR project on the function of CRISPR-Cas systems in bacteria has led to the development of powerful high-throughput screens. These screens have provided important novel insights into the genetics of the Escherichia coli species. In addition, this project has established novel strategies and tools that we have shared with the broader scientific community. These tools are now accelerating the pace of research in bacterial genetics and how they can impact our health.
The objective of this project was to shed light on the genetic requirements and molecular consequences of DNA cleavage by CRISPR-Cas systems and in particular the interplay between CRISPR and DNA repair pathways. The knowledge gained was then be applied to establish new methods for the manipulation of bacterial genomes using CRISPR and to enable high-throughput screens using CRISPR libraries.
The first aim was to study of the coevolution and interplay between CRISPR immunity and DNA repair pathways. CRISPR-Cas systems introduce double strand breaks into DNA of invading genetic material and use DNA fragments to acquire novel spacers during adaptation. Double strand breaks are the substrate of several bacterial DNA repair pathways, paving the way for interactions between them and CRISPR-Cas systems. We identified computationally several such interactions and investigated experimentally one of them that could have relevance for genome editing applications (Bernheim, 2017; Bernheim, 2019). Our findings further contribute to explain the scattered distribution of CRISPR-Cas systems in bacterial genomes.
The second aim of the proposal was to determine the genetic requirements for efficient CRISPR targeting. We have conducted an in-depth study of Cas9 binding to target genes and how this binding is affected by mismatches between the guide RNA and the target (Vigouroux, 2018). The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to silence target. 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 robustly reduce gene expression by defined relative amounts and have demonstrated the broad applicability of this method to the study of genetic regulation and cellular physiology. We further established methods for the control of gene expression in E. coli and S. aureus that can be conveniently used by microbiologist (Depardieu, Methods, 2019). We further improved our understanding of the sequence determinants of Cas9 activity in bacteria by combining high throughput screening data and machine learning approaches (Calvo-Villamañán, NAR, 2020). With this last piece of work, we provide design tools for researchers as well as guide RNA libraries to conduct powerful high throughput screens available at crispr.pasteur.fr .
The third aim of the proposal was to investigate the toxicity of Cas9 in bacteria, and more specifically the mechanism underlying the toxicity linked to specific sequence motifs in the guide RNA (Cui, 2018). We have now demonstrated that this phenomenon is due to off-target binding of Cas9 to the promoter of genes with as little as 4nt of complementarity. In some circumstances, this binding can lead to the silencing of the off-target gene, and toxicity when the gene is essential. A manuscript describing these findings is in preparation.
The fourth aim of the proposal was to develop high-throughput screens using CRISPR libraries in bacteria. High throughput CRISPR-Cas9 screens have recently emerged as a powerful tool to decipher gene functions and genetic interactions. We used a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcriptions in E. coli (Cui, 2018). This enabled us to identify the design rules for gene silencing using dCas9 in E. coli. This knowledge was used in subsequent studies to design improved libraries (Calvo-Villamañán, NAR, 2020) and investigate different aspects of the genetics of E. coli. In particular we used our approach to investigate bacteriophage host factors (Rousset, 2018, Plos Genetics), as well as how accessory genes impact gene essentiality in the E. coli species (Rousset, 2021). Insights obtained from this study further led to the unexpected discovery of novel anti-phage defense systems carried prophages and satellites of phages within the E. coli gene, some of which modify the essentiality of core genes (Rousset, 2022).
Altogether the project has contributed to improving our understanding of CRISPR-Cas systems in bacteria and their interaction with DNA repair pathways, and how Cas9 binding to DNA can silence gene expression. It has also led to the development of novel methods of investigation for bacterial genetics that we have shared with the community and which are already being adopted by labs around the world.
The first aim was to study of the coevolution and interplay between CRISPR immunity and DNA repair pathways. CRISPR-Cas systems introduce double strand breaks into DNA of invading genetic material and use DNA fragments to acquire novel spacers during adaptation. Double strand breaks are the substrate of several bacterial DNA repair pathways, paving the way for interactions between them and CRISPR-Cas systems. We identified computationally several such interactions and investigated experimentally one of them that could have relevance for genome editing applications (Bernheim, 2017; Bernheim, 2019). Our findings further contribute to explain the scattered distribution of CRISPR-Cas systems in bacterial genomes.
The second aim of the proposal was to determine the genetic requirements for efficient CRISPR targeting. We have conducted an in-depth study of Cas9 binding to target genes and how this binding is affected by mismatches between the guide RNA and the target (Vigouroux, 2018). The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to silence target. 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 robustly reduce gene expression by defined relative amounts and have demonstrated the broad applicability of this method to the study of genetic regulation and cellular physiology. We further established methods for the control of gene expression in E. coli and S. aureus that can be conveniently used by microbiologist (Depardieu, Methods, 2019). We further improved our understanding of the sequence determinants of Cas9 activity in bacteria by combining high throughput screening data and machine learning approaches (Calvo-Villamañán, NAR, 2020). With this last piece of work, we provide design tools for researchers as well as guide RNA libraries to conduct powerful high throughput screens available at crispr.pasteur.fr .
The third aim of the proposal was to investigate the toxicity of Cas9 in bacteria, and more specifically the mechanism underlying the toxicity linked to specific sequence motifs in the guide RNA (Cui, 2018). We have now demonstrated that this phenomenon is due to off-target binding of Cas9 to the promoter of genes with as little as 4nt of complementarity. In some circumstances, this binding can lead to the silencing of the off-target gene, and toxicity when the gene is essential. A manuscript describing these findings is in preparation.
The fourth aim of the proposal was to develop high-throughput screens using CRISPR libraries in bacteria. High throughput CRISPR-Cas9 screens have recently emerged as a powerful tool to decipher gene functions and genetic interactions. We used a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcriptions in E. coli (Cui, 2018). This enabled us to identify the design rules for gene silencing using dCas9 in E. coli. This knowledge was used in subsequent studies to design improved libraries (Calvo-Villamañán, NAR, 2020) and investigate different aspects of the genetics of E. coli. In particular we used our approach to investigate bacteriophage host factors (Rousset, 2018, Plos Genetics), as well as how accessory genes impact gene essentiality in the E. coli species (Rousset, 2021). Insights obtained from this study further led to the unexpected discovery of novel anti-phage defense systems carried prophages and satellites of phages within the E. coli gene, some of which modify the essentiality of core genes (Rousset, 2022).
Altogether the project has contributed to improving our understanding of CRISPR-Cas systems in bacteria and their interaction with DNA repair pathways, and how Cas9 binding to DNA can silence gene expression. It has also led to the development of novel methods of investigation for bacterial genetics that we have shared with the community and which are already being adopted by labs around the world.
Our achievements have significantly advanced CRISPR-Cas technologies applied to bacteria, as well as yielded significant novel insights into the genetics of the E. coli species. The tools and methods we developed are currently being used by a large number of laboratories around the world, with CRISPRi quickly becoming a standard approach of investigation in bacterial genetics. The project has also led to unexpected findings, including the toxicity of guide RNAs sharing specific sequence motifs (Cui, 2018). We have now solved the mechanism behind this mysterious phenomenon and a manuscript is in preparation. Another notable unexpected finding was how the identification of how anti-phage defense systems can modify the essentiality of core bacterial genes. This finding described in (Rousset, 2021), led to a follow up study revealing a large number of novel anti-phage defense systems carried by prophages and satellites of phages (Rousset, 2022).