Project description:Enzymes maintain metabolism and their concentration affects cellular fitness. High enzyme-levels are costly, but low enzyme-levels can limit metabolic flux. Here, we used CRISPR interference (CRISPRi) to study the consequences of decreasing metabolic enzymes in E. coli below wild-type levels. A time-resolved competition assay with a metabolism-wide CRISPRi library showed that fitness defects appeared late after induction of knockdowns. This suggested that metabolism is robust against decreases of enzymes. The metabolome and proteome of 30 CRISPRi strains revealed the mechanisms that enabled this robustness. First, substrates and allosteric effectors buffered knockdowns by increasing the activity of target-enzymes. Later, metabolite-transcription interactions compensated knockdowns by upregulating the target-pathway or bypass-pathways. For example, we found a new regulation strategy in which 6-phosphogluconate is responsible for bypassing bottlenecks in the pentose-p pathway via the Entner-Doudoroff-pathway. Thus, regulatory metabolites buffer decreases of enzyme-levels, which can occur in nature due to expression noise, mutations or environmental conditions.
Project description:CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knockdown gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus. Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically-dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter. We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in Caulobacter.
Project description:we performed lentiviral CRISPR interference (CRISPRi) by recruiting dCas9 fused with the KRAB domain to the CSMD1 enhancer (fam3) in the neuronal precursor cell line – Lund human mesencephalic (LUHMES). Given that the expression of CSMD1 was not detectable in LUHMES cells we differentiated these cells into neurons. Differentiated neurons with CRISPRi of CSMD1 enhancer showed significantly higher expression of CSMD1 than control.
Project description:Assess the on- and off-target effects of dox-inducible CRISPR/Cas9 and CRISPRi constructs in a human iPS cell line. Transcript quantification of 3 cell lines, each plus or minus doxycycline and with or without specific single guide RNAs (sgRNAs), with 2 biological replicates each.
Project description:We performed RNA-seq to examine RNA expression profiles during MCF10A-ER-Src cell transformation and upon knockdowns of transcription factors
Project description:We develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell. We perform ATAC-seq on CRISPRi perturbed Jurkat T cells to investigate chromatin accessibility changes upon perturbation of individual regulatory elements.
Project description:CRISPR interference (CRISPRi) genetic screens use programmable repression of gene expression to systematically explore questions in cell biology and genetics. However, wider adoption of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and lack of consensus on the choice of CRISPRi effector proteins. Here, we address these challenges to present next-generation CRISPRi sgRNA libraries and effectors. First, we combine empiric sgRNA selection with a dual sgRNA library design to generate an ultra-compact, highly active CRISPRi sgRNA library. Next, we rigorously compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an optimal balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines which stably express Zim3-dCas9 and demonstrate robust on-target knockdown across these cell lines. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.
