The RNA-guided Cas9 nuclease from CRISPR-Cas systems has emerged as a powerful biotechnological tool. The specificity of Cas9 can be reprogrammed to cleave desired sequences in a cell's chromosome simply by changing the sequence of a small guide RNA. Unlike in most eukaryotes, Cas9 cleavage in the chromosome of bacteria has been reported to kill the cell. However, the mechanism of cell death remains to be investigated. Bacteria mainly rely on homologous recombination (HR) with sister chromosomes to repair double strand breaks. Here, we show that the simultaneous cleavage of all copies of the Escherichia coli chromosome at the same position cannot be repaired, leading to cell death. However, inefficient cleavage can be tolerated through continuous repair by the HR pathway. In order to kill cells reliably, HR can be blocked using the Mu phage Gam protein. Finally, the introduction of the non-homologous end joining (NHEJ) pathway from Mycobacterium tuberculosis was not able to rescue the cells from Cas9-mediated killing, but did introduce small deletions at a low frequency. This work provides a better understanding of the consequences of Cas9 cleavage in bacterial chromosomes which will be instrumental in the development of future CRISPR tools.
High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. Here we use a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcription in Escherichia coli. Using a machine-learning approach, we reveal that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence. This effect occurs at high dCas9 concentrations and can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression. Our results also highlight the fact that off-targets with as little as nine nucleotides of homology to the guide RNA can strongly block gene expression. Altogether this study provides important design rules to safely use dCas9 in E. coli.
We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.
High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E. coli during growth in rich medium. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential. In addition, we took advantage of the intermediate repression levels obtained when targeting the template strand of genes to show that cells are very sensitive to the expression level of a limited set of essential genes. Our data can be visualized on CRISPRbrowser, a custom web interface available at crispr.pasteur.fr. We then apply the screen to discover E. coli genes required by phages λ, T4 and 186 to kill their host, highlighting the involvement of diverse host pathways in the infection process of the three tested phages. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. Finally, using a plasmid packaging system and a transduction assay, we identify genes required for the formation of functional λ capsids, thus covering the entire phage cycle. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics.
Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase “kicks out” dCas9 from the target and completes transcription. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. Alternatively, tuning repression by changing dCas9 concentration is noisy and promoter‐strength dependent. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto‐repressor. Second, we study the impact of amount variations of cell‐wall synthesizing enzymes on cell morphology. Finally, we multiplex the system to obtain any combination of fractional repression of two genes.
Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to longrange DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250-to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5-to 10-kb distances, was able to increase contact probability 10-to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5-to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells. j factor | TPM | promoter-enhancer I nteractions between proteins bound to separate sites on the same DNA molecule are critical in gene regulation and other DNA processes (1-4). The DNA separation between functionally interacting sites ranges from a few base pairs to hundreds of kilobase pairs, as in the case of some eukaryotic enhancers and their promoters. At short separations, it is clear that the DNA acts as a tether that keeps one site in the vicinity of the other so that the proteins at one site can find the other site in 3D space more efficiently than if they were free in solution (Fig. 1). Tethering is also a way to provide specificity because it aids interaction with linked sites but not unlinked sites. However, as the separation between the sites increases, this tethering effect becomes weaker, and it is not understood how the DNA linkage between widely separated sites, for example, between enhancers and promoters, provides the efficiency and specificity required for proper regulation.The effect of DNA tethering can be quantified by the factor j LOOP (M), the effective molar concentration of one site on the DNA relative to the other, or as the free energy of the DNA looping reaction ΔG LOOP (Fig. 1) (5-9). These parameters are interconvertible: ΔG LOOP = -RTlnj LOOP (kcal/mol; the reference j is 1 M). The formation of a naked DNA loop is in itself an energetically unfavorable reaction (ΔG LOOP is positive) under physiological conditions due to the enthalpic cost of DNA bending and twisting (particularly important for s...
How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL, is known already to enhance repression at the lysogenic promoter PRM, located at OR. Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL. Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α-C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer-promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements. I n eukaryotes, initiation of transcription is often regulated by enhancers, DNA sequences that can be located many kilobases away from the promoter. Enhancers are usually complex, being composed of multiple DNA elements that bind a variety of proteins (1, 2). The enhancer and the promoter are brought into close contact, looping the intervening DNA (3, 4), thus placing these binding elements close to the promoter. However, due to the lack of detailed understanding of enhancer-promoter complexes, how enhancers efficiently and specifically regulate initiation remains obscure (5).The action of DNA elements at and around a promoter is easier to understand. These sequences can be envisaged as a local scaffold for the assembly of transcription-favoring or transcription-inhibiting protein complexes. The promoter elements bind the transcription machinery, and promoter-proximal DNA elements position other proteins so that they can interact, favorably or unfavorably, with this machinery (2, 6-9). In prokaryotes, these promoter complexes are relatively simple. Repression of transcription is usually achieved by proteins positioned to compete with binding of RNA polymerase (RNAP), whereas transcription can be activated by proteins positioned adjacent to RNAP that contact it to stabilize initiation intermediates (8,10,11).The CI repressor of bacteriophage λ has been seminal in understanding how promoter-proximal elements are used in transcriptional regulation. Two CI binding sites, OR1 and OR2, overlap the lytic PR promoter and cooperative binding of two CI dimers to these sites competes with RNAP binding and represses the promoter. Extensive study of this complex has culminated in crystal structures that reveal how CI binds to the DNA a...
Type II CRISPR-Cas systems introduce double-strand breaks into DNA of invading genetic material and use DNA fragments to acquire novel spacers during adaptation. These breaks can be the substrate of several DNA repair pathways, paving the way for interactions. We report that non-homologous end-joining (NHEJ) and type II-A CRISPR-Cas systems only co-occur once among 5563 fully sequenced prokaryotic genomes. We investigated experimentally the possible molecular interactions using the NHEJ pathway from Bacillus subtilis and the type II-A CRISPR-Cas systems from Streptococcus thermophilus and Streptococcus pyogenes. Our results suggest that the NHEJ system has no effect on CRISPR immunity. On the other hand, we provide evidence for the inhibition of NHEJ repair by the Csn2 protein. Our findings give insights on the complex interactions between CRISPR-Cas systems and repair mechanisms in bacteria, contributing to explain the scattered distribution of CRISPR-Cas systems in bacterial genome.
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