Functional interrogation of non-coding DNA through CRISPR genome editing

Canver, Matthew C.; Bauer, Daniel E.; Orkin, Stuart H.

doi:10.1016/j.ymeth.2017.03.008

Cited by 27 publications

(19 citation statements)

References 211 publications

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“…High-throughput gene knockdown or knockout screens have also become a popular experimental approach to identify genes that are important for a phenotype of interest. These screens can be implemented with short hairpin RNA or CRISPR-Cas9 genetic perturbation to systematically test which genes or regulatory elements are essential for a phenotype, such as hematopoietic lineage differentiation (Canver et al, 2017;Nandakumar et al, 2019). GWAS of blood cell traits, combined with these functional analyses, have provided tremendous value for understanding the transcriptional regulatory mechanisms underlying hematopoiesis.…”

Section: Population-based Genetic Studies Of Hematopoiesis and Their mentioning

confidence: 99%

The genetics of human hematopoiesis and its disruption in disease

2019

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Hematopoiesis, or the process of blood cell production, is a paradigm of multi‐lineage cellular differentiation that has been extensively studied, yet in many aspects remains incompletely understood. Nearly all clinically measured hematopoietic traits exhibit extensive variation and are highly heritable, underscoring the importance of genetic variation in these processes. This review explores how human genetics have illuminated our understanding of hematopoiesis in health and disease. The study of rare mutations in blood and immune disorders has elucidated novel roles for regulators of hematopoiesis and uncovered numerous important molecular pathways, as seen through examples such as Diamond‐Blackfan anemia and the GATA2 deficiency syndromes. Additionally, population studies of common genetic variation have revealed mechanisms by which human hematopoiesis can be modulated. We discuss advances in functionally characterizing common variants associated with blood cell traits and discuss therapeutic insights, such as the discovery of BCL11A as a modulator of fetal hemoglobin expression. Finally, as genetic techniques continue to evolve, we discuss the prospects, challenges, and unanswered questions that lie ahead in this burgeoning field.

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Section: Population-based Genetic Studies Of Hematopoiesis and Their mentioning

confidence: 99%

The genetics of human hematopoiesis and its disruption in disease

2019

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“…This protocol has been used in previously published works 11,12 . CRISPR mutagenesis allows for the study of both coding and non-coding regions of the genome 13 . This involves usage of one sgRNA for arrayed experiments or multiple sgRNAs for pooled experiments.…”

Section: Applications Of the Methods And Development Of The Protocolmentioning

confidence: 99%

“…Select ~10-20 colonies from the dilution plates (from Step 37) for screening by mini-scale plasmid preparation and subsequent Sanger sequencing (as in to determine if the sgRNA library has been PCR amplified. Given that the oligonucleotides for multiple libraries can be synthesized on the same programmable microarray (Steps[13][14]…”

mentioning

confidence: 99%

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome editing experiments

Canver

Haeussler

Bauer

et al. 2017

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CRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.

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“…This protocol has been used in previously published works to functionally interrogate the BCL11A enhancer as well as to evaluate potential functional sequences within all DNase hypersensitive sites (DHSs) in proximity to the MYB gene 11,12 . CRISPR mutagenesis allows for the study of both coding and noncoding regions of the genome 13 . This involves usage of one sgRNA for arrayed experiments or multiple sgRNAs for pooled experiments.…”

Section: Introductionmentioning

confidence: 99%

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

et al. 2018

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CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNARNARNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4–5 weeks by non-experts, as well as computational data analysis that can be performed in 1–2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

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Functional interrogation of non-coding DNA through CRISPR genome editing

Cited by 27 publications

References 211 publications

The genetics of human hematopoiesis and its disruption in disease

The genetics of human hematopoiesis and its disruption in disease

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome editing experiments

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

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