Topical Collection "Gene Editing"

Editors

Dr. Philip Hublitz
E-Mail Website
Collection Editor
Genome Engineering Facility, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK
Interests: mouse models; recombineering technology; CRISPR/Cas9 technology; Cas9-screens; dCas9-imaging; general regulation of transcription and epigenesis
Special Issues, Collections and Topics in MDPI journals
Dr. Antónia Monteiro
E-Mail Website
Collection Editor
1. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
2. Yale-NUS College, 10 College Avenue West, Singapore 138609, Singapore
Interests: butterfly wing patterns; evo-devo; functional genetics; plasticity; eyespots; evolution; sexual and natural selection; behaviour
Special Issues, Collections and Topics in MDPI journals

Topical Collection Information

Dear colleagues,

With the emergence of CRISPR/Cas9, an unprecedented variety of novel approaches and possibilities for genomic engineering are unveiled on a regular basis. These developments are accelerating the pace of research in model systems, as well as creating a plethora of emerging model systems invaluable for comparative functional approaches across the tree of life. The CRISPR/Cas9 toolbox has already allowed the manipulation of gene function in basal land plants, butterflies, crickets and Atlantic salmon, and many more are sure to follow.

This topical collection on “Gene Editing” aims to provide a forum for discussions on the latest technical developments in the fields of general genome engineering technologies, including (i) the establishment of cell culture systems, and (ii) the development of established and emerging organismal models by CRISPR/Cas9 or similar genome engineering tools. A constant flow of reports demonstrates the continuous refinement of the CRISPR/Cas9 revolutionary tool. Invariably, however, new challenges or missing details in the implementation of this technology become apparent when applied to specific model systems, which need to be addressed. Our topical collection aims to keep up with the most recent developments, refinements, and latest achievements in gene editing available today. We encourage contributions from laboratories working at the forefront of the development of novel CRISPR/Cas9 approaches, with the goal of sharing these details and accelerating the speed of functional genetic discovery across the tree of life, in well-established as well as in emerging model systems.

Dr. Philip Hublitz
Dr. Antónia Monteiro
Collection Editors

Manuscript Submission Information

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Keywords

  • CRISPR/Cas9
  • Genome engineering
  • Gene editing
  • Established and emerging model systems
  • Model generation
  • Functional genetics using KI and KO
  • Spatiotemporal regulation
  • Arrayed and pooled sgRNA-Screening
  • Cas9 efficiency and fidelity
  • New tools in the CRISPR/Cas9 toolbox

Published Papers (6 papers)

2021

Jump to: 2018

Technical Note
In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells
Methods Protoc. 2021, 4(2), 40; https://doi.org/10.3390/mps4020040 - 08 Jun 2021
Cited by 3 | Viewed by 1465
Abstract
Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the [...] Read more.
Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens. Full article
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Figure 1

2018

Jump to: 2021

Protocol
High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates
Methods Protoc. 2018, 1(3), 29; https://doi.org/10.3390/mps1030029 - 01 Aug 2018
Cited by 2 | Viewed by 4017
Abstract
The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array [...] Read more.
The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis. Full article
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Figure 1

Protocol
Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
Methods Protoc. 2018, 1(3), 28; https://doi.org/10.3390/mps1030028 - 30 Jul 2018
Cited by 14 | Viewed by 5081
Abstract
The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells [...] Read more.
The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4–7 fold higher than starting cell numbers within 9–12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations. Full article
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Protocol
A Two-Step Method for Obtaining Highly Pure Cas9 Nuclease for Genome Editing, Biophysical, and Structural Studies
Methods Protoc. 2018, 1(2), 17; https://doi.org/10.3390/mps1020017 - 30 May 2018
Cited by 6 | Viewed by 3461
Abstract
Cas9 is a site-specific RNA-guided endonuclease (RGEN) that can be used for precise genome editing in various cell types from multiple species. Ribonucleoprotein (RNP) complexes, which contains the Cas9 protein in complex with a guide RNA, are sufficient for the precise editing of [...] Read more.
Cas9 is a site-specific RNA-guided endonuclease (RGEN) that can be used for precise genome editing in various cell types from multiple species. Ribonucleoprotein (RNP) complexes, which contains the Cas9 protein in complex with a guide RNA, are sufficient for the precise editing of genomes in various cells. This DNA-free method is more specific in editing the target sites and there is no integration of foreign DNA into the genome. Also, there are ongoing studies into the interactions of Cas9 protein with modified guide RNAs, as well as structure-activity studies of Cas9 protein and its variants. All these investigations require highly pure Cas9 protein. A single-step metal affinity enrichment yielding impure Cas9 is the most common method of purification described. This is sufficient for many gene editing applications of this protein. However, to obtain Cas9 of higher purity, which might be essential for biophysical characterization, chemical modifications, and structural investigations, laborious multi-step protocols are employed. Here, we describe a two-step Cas9 purification protocol that uses metal affinity enrichment followed by cation exchange chromatography. This simple method can yield a milligram of highly pure Cas9 protein per liter of culture in a single day. Full article
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Protocol
CRISPR-Cas9 Mediated Genome Editing in Bicyclus anynana Butterflies
Methods Protoc. 2018, 1(2), 16; https://doi.org/10.3390/mps1020016 - 15 May 2018
Cited by 7 | Viewed by 5010
Abstract
CRISPR-Cas9 is revolutionizing the field of genome editing in non-model organisms. The robustness, ease of use, replicability and affordability of the technology has resulted in its widespread adoption among researchers. The African butterfly Bicyclus anynana is an emerging model lepidopteran species in the [...] Read more.
CRISPR-Cas9 is revolutionizing the field of genome editing in non-model organisms. The robustness, ease of use, replicability and affordability of the technology has resulted in its widespread adoption among researchers. The African butterfly Bicyclus anynana is an emerging model lepidopteran species in the field of evo-devo, with a sequenced genome and amenable to germ line transformation. However, efficient genome editing tools to accelerate the pace of functional genetic research in this species have only recently become available with CRISPR-Cas9 technology. Here, we provide a detailed explanation of the CRISPR-Cas9 protocol we follow in the lab. The technique has been successfully implemented to knock-out genes associated with eyespot development and melanin pigmentation. Full article
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Graphical abstract

Protocol
Generating CRISPR/Cas9-Derived Mutant Mice by Zygote Cytoplasmic Injection Using an Automatic Microinjector
Methods Protoc. 2018, 1(1), 5; https://doi.org/10.3390/mps1010005 - 12 Jan 2018
Cited by 6 | Viewed by 5340
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of [...] Read more.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of producing materials in comparison to other methodologies (such as embryonic stem cells, ESCs) and the ability to simultaneously target multiple genes in one injection session. Here we describe a step by step protocol, from preparation of materials through to injection and validation of a cytoplasmic injection, which can be used to generate CRISPR mutants. This can be accomplished from start of injection to completion within 2–4 h with high survival and developmental rates of injected zygotes and offers significant advantages over pronuclear and other previously described methodologies for microinjection. Full article
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Figure 1

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