Identifying Novel Enhancer Elements with CRISPR-Based Screens

Klein, Jason C.; Chen, Wei; Gasperini, Molly; Shendure, Jay

doi:10.1021/acschembio.7b00778

Cited by 27 publications

(19 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For many if not most applications of CRISPR/Cas9-mediated genome engineering, it is used in conjunction with the cell's endogenous NHEJ machinery to introduce short indels in a targeted fashion (Mali et al 2013;Cong et al 2013;Jinek et al 2013) , e.g. to disrupt the function of genes or regulatory elements (Wang et al 2015;Gasperini et al 2017;Klein et al 2018) or to introduce irreversible changes that record cell lineage or molecular events (McKenna et al 2016;Frieda et al 2017;Kalhor et al 2018) . However, despite NHEJ's central importance to this transformative tool, our understanding of the processes that determine the rate and patterns of NHEJ-mediated errors remains incomplete.…”

Section: Introductionmentioning

confidence: 99%

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

Chen

McKenna

Schreiber

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled~1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6,872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: 1) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, 2) deletions are predominantly associated with microhomology, and 3) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.

show abstract

Section: Introductionmentioning

confidence: 99%

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

Chen

McKenna

Schreiber

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Alternative methods of assigning the target genes of regulatory regions, such as those based on correlated chromatin activity of promoters and enhancers (20,(93)(94)(95)(96) may account for the effects of these regions. Emerging high-throughput functional screens of cis-regulatory activity (8,9,64,97) are proving to be instrumental for direct functional validation of enhancer targets identified with either approach.…”

Section: Discussionmentioning

confidence: 99%

Functional effects of variation in transcription factor binding highlight long-range gene regulation by epromoters

Mitchelmore

Grinberg

Wallace

et al. 2019

Preprint

View full text Add to dashboard Cite

Identifying DNA cis-regulatory modules (CRMs) that control the expression of specific genes is crucial for deciphering the logic of transcriptional control. Natural genetic variation can point to the possible gene regulatory function of specific sequences through their allelic associations with gene expression. However, comprehensive identification of causal regulatory sequences in bruteforce association testing without incorporating prior knowledge is challenging due to limited statistical power and effects of linkage disequilibrium. Sequence variants affecting transcription factor (TF) binding at CRMs have a strong potential to influence gene regulatory function, which provides a motivation for prioritising such variants in association testing. Here, we generate an atlas of CRMs showing predicted allelic variation in TF binding affinity in human lymphoblastoid cell lines (LCLs) and test their association with the expression of their putative target genes inferred from Promoter Capture Hi-C and immediate linear proximity. We reveal over 1300 CRM TF-binding variants associated with target gene expression, the majority of them undetected with standard association testing. A large proportion of CRMs showing associations with the expression of genes they contact in 3D localise to the promoter regions of other genes, supporting the notion of 'epromoters': dual-action CRMs with promoter and distal enhancer activity.

show abstract

“…Unlike RNAi, CRISPR/Cas9-based genome disruption can interrogate not only the function of transcriptionally active regions of the genome but also the non-coding portions, such as enhancers. Several high-throughput strategies have been established to identify possible enhancer regions through CRISPR/Cas9-based genome disruption, many of which use a tiling approach in a cultured cell setting and comprehensively survey a locus of interest with a collection of short guide RNAs (sgRNAs) designed to cover the entirety of the locus (reviewed in Catarino and Stark, 2018;Klein et al, 2018;Lopes et al, 2016). More recently, the next generation of CRISPR/Cas9 technologies, such as CRISPR-based transcriptional activation (CRISPRa) or interference (CRISPRi), have allowed researchers to manipulate the transcription of endogenous loci by taking advantage of the sequence specificity of the CRISPR/Cas9 system (reviewed in Adli, 2018;Pickar-Oliver and Gersbach, 2019).…”

Section: Crispr/cas9-based Screeningmentioning

confidence: 99%

How to study enhancers in non-traditional insect models

Tomoyasu

Halfon

2020

Journal of Experimental Biology

View full text Add to dashboard Cite

Transcriptional enhancers are central to the function and evolution of genes and gene regulation. At the organismal level, enhancers play a crucial role in coordinating tissue-and context-dependent gene expression. At the population level, changes in enhancers are thought to be a major driving force that facilitates evolution of diverse traits. An amazing array of diverse traits seen in insect morphology, physiology and behavior has been the subject of research for centuries. Although enhancer studies in insects outside of Drosophila have been limited, recent advances in functional genomic approaches have begun to make such studies possible in an increasing selection of insect species. Here, instead of comprehensively reviewing currently available technologies for enhancer studies in established model organisms such as Drosophila, we focus on a subset of computational and experimental approaches that are likely applicable to non-Drosophila insects, and discuss the pros and cons of each approach. We discuss the importance of validating enhancer function and evaluate several possible validation methods, such as reporter assays and genome editing. Key points and potential pitfalls when establishing a reporter assay system in non-traditional insect models are also discussed. We close with a discussion of how to advance enhancer studies in insects, both by improving computational approaches and by expanding the genetic toolbox in various insects. Through these discussions, this Review provides a conceptual framework for studying the function and evolution of enhancers in non-traditional insect models.

show abstract

Identifying Novel Enhancer Elements with CRISPR-Based Screens

Cited by 27 publications

References 60 publications

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

Functional effects of variation in transcription factor binding highlight long-range gene regulation by epromoters

How to study enhancers in non-traditional insect models

Contact Info

Product

Resources

About