Lun Cui

One-Step Cloning and Chromosomal Integration of DNA

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.

Consequences of Cas9 cleavage in the chromosome of Escherichia coli

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 cells 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.

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors.

Significance Proteins bound to DNA often interact with proteins bound elsewhere on the same DNA to regulate gene expression. The intervening DNA tethers the proteins near each other, making their interaction efficient and specific, but the importance of this tethering effect is poorly understood at large DNA separations. We quantitated tethering inside bacterial cells, using two different proteins at separations up to 10,000 bp, to show that tethering is strong enough to drive efficient interactions over these distances. The same interactions were ∼10-fold weaker outside cells, implying that cellular factors enhance tethering. However, tethering was lost at a DNA separation of 500,000 bp inside bacteria, indicating special mechanisms inside eukaryotic cells to provide efficient and specific interactions over such distances. 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 long-range 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.

Enhancer-like long-range transcriptional activation by λ CI-mediated DNA looping.

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.

A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9

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.CRISPR interference (CRISPRi) is a method for targeted silencing of transcription that requires the coexpression of protein dCas9 and a customized guide RNA. Here, Cui et al. show that certain guide RNAs induce toxicity in E. coli, and provide design rules to minimize off-target effects.

Inhibition of NHEJ repair by type II-A CRISPR-Cas systems in bacteria

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.The double-strand breaks generated by CRISPR-Cas systems are the target of multiple DNA repair pathways. Here the authors find incompatibility between NHEJ and type II-A CRISPR-Cas systems due to Csn2 mediated inhibition of end-joining.

Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes

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.

Bacteriophage lambda repressor mediates the formation of a complex enhancer-like structure.

For the past 40 years, bacteriophage lambda has been crucial in revealing fundamental principles underlying control of transcription by elements positioned close to promoters. With the discovery that lambda CI repressors bound to distant sites can interact efficiently, lambda also provides a model for long range gene regulation, including the action of enhancer elements.

Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors

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 pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome of E. coli. We then apply the screen to discover E. coli genes required by phages λ, T4 and 186 to kill their host. In particular, we show that colanic acid capsule is a barrier to all three phages. Finally, cloning the library on a plasmid that can be packaged by λ enables to identify genes required for the formation of functional λ capsids. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria in order to identify genes linked to a given phenotype.

Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

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 dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. 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 copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.