Project description:A critical goal in functional genomicsis evaluating which non-coding elements contribute togene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach is valuable for elucidating functional transcription factor binding motifs or other repeated genomic sequences and is easily implementable with existing tools.
Project description:A critical goal in functional genomicsis evaluating which non-coding elements contribute togene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach is valuable for elucidating functional transcription factor binding motifs or other repeated genomic sequences and is easily implementable with existing tools.
Project description:CRISPR interference (CRISPRi), the targeting of a catalytically dead Cas protein to block transcription, is the leading technique to silence gene expression in bacteria. However, design rules for CRISPRi remain poorly defined, limiting predictable design for gene interrogation, pathway manipulation, and high-throughput screens. Here we develop a best-in-class prediction algorithm for guide silencing efficiency by systematically investigating factors influencing guide depletion in multiple genome-wide essentiality screens, with the surprising discovery that gene-specific features such as transcriptional activity substantially impact prediction of guide activity. Accounting for these features as part of algorithm development allowed us to develop a mixed-effect random forest regression model that provides better estimates of guide efficiency than existing methods, as demonstrated in an independent saturating screen. We further applied methods from explainable AI to extract interpretable design rules from the model, such as sequence preferences in the vicinity of the PAM distinct from those previously described for genome engineering applications. Our approach provides a blueprint for the development of predictive models for CRISPR technologies where only indirect measurements of guide activity are available.
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:RNA-seq was performed to identify genes affected by CRISPRi (dCas9-KRAB) with sgRNA (sgSFTPB#2) targeting the first intronic region of human SFTPB bound by NKX2-1 in an unbiased genome-wide fashion using A549 lung carcinoma cells that constitutively express dCas9-KRAB and NKX2-1.
Project description:The Mobile CRISPRi system with and without mRFP-targeting sgRNA was engineered into Pseudomonas aeruginosa PA14 strain with chromosomally encoded mRFP. RNA was isolated from these strains, and the corresponding cDNA library was synthesized and sequenced in 150 bp paired-end reads. Approximately 1,000,000 reads were collected for each of the two samples, with ~94% alignment to PA14 WT by Bowtie254, and transcripts were counted with HTSeq55. Only genes with a non-normalized read count greater than 1 in both samples were included in analysis, with a coverage of 1286 genes (~20% genome). This data shows that the Mobile CRISPRi system is selective for sgRNA-guided knockdown of mRFP.