Methods and Protocols

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28 pages, 1262 KiB

Open AccessFeature PaperReview

CRISPR-Based Editing Techniques for Genetic Manipulation of Primary T Cells

by Mateusz Kotowski and Sumana Sharma

Methods Protoc. 2020, 3(4), 79; https://doi.org/10.3390/mps3040079 - 18 Nov 2020

Cited by 9 | Viewed by 4523

Abstract

While clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing techniques have been widely adapted for use in immortalised immune cells, efficient manipulation of primary T cells has proved to be more challenging. Nonetheless, the rapid expansion of the CRISPR toolbox accompanied by [...] Read more.

While clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing techniques have been widely adapted for use in immortalised immune cells, efficient manipulation of primary T cells has proved to be more challenging. Nonetheless, the rapid expansion of the CRISPR toolbox accompanied by the development of techniques for delivery of CRISPR components into primary T cells now affords the possibility to genetically manipulate primary T cells both with precision and at scale. Here, we review the key features of the techniques for primary T cell editing and discuss how the new generation of CRISPR-based tools may advance genetic engineering of these immune cells. This improved ability to genetically manipulate primary T cells will further enhance our fundamental understanding of cellular signalling and transcriptional networks in T cells and more importantly has the potential to revolutionise T cell-based therapies. Full article

(This article belongs to the Special Issue Advances of CRISPR-Cas Systems for Genome Engineering)

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Other

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14 pages, 4845 KiB

Open AccessProtocol

An Efficient Workflow for Screening and Stabilizing CRISPR/Cas9-Mediated Mutant Lines in Bombyx mori

by Daniel Brady, Alessio Saviane, Silvia Cappellozza and Federica Sandrelli

Methods Protoc. 2021, 4(1), 4; https://doi.org/10.3390/mps4010004 - 29 Dec 2020

Cited by 7 | Viewed by 3681

Abstract

The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic [...] Read more.

The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. mori lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects. Full article

(This article belongs to the Special Issue Advances of CRISPR-Cas Systems for Genome Engineering)

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11 pages, 858 KiB

Open AccessTechnical Note

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

by Joseph Blayney, Evangeline M. Foster, Marta Jagielowicz, Mira Kreuzer, Matteo Morotti, Katharina Reglinski, Julie Huiyuan Xiao and Philip Hublitz

Methods Protoc. 2020, 3(3), 53; https://doi.org/10.3390/mps3030053 - 29 Jul 2020

Cited by 6 | Viewed by 3590

Abstract

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have [...] Read more.

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions. Full article

(This article belongs to the Special Issue Advances of CRISPR-Cas Systems for Genome Engineering)

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