Guidelines for optimized gene knockout using CRISPR/Cas9

Campenhout, Claude Van; Cabochette, Pauline; Veillard, Anne-Clémence; Laczik, Miklos; Zelisko-Schmidt, Agnieszka; Sabatel, Céline; Dhainaut, Maxime; Vanhollebeke, Benoît; Gueydan, Cyril; Kruys, Véronique

doi:10.2144/btn-2018-0187

Cited by 37 publications

(22 citation statements)

References 77 publications

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“…The CRISPR/Cas9 gene editing has evolved as the most powerful tool for efficient and specific genome engineering to create genetic models for both fundamental research and crop genetic improvements although this new technology is being developed rapidly (Shan et al, 2013;Van Campenhout et al, 2019;Wolter et al, 2019). This gene editing system has been successfully used to generate a broad range of stable mutagenesis at specific locus in several important crops such as rice (Zhang et al, 2014), wheat , maize (Svitashev et al, 2015), soybean , tomato (Ito and Nakano, 2015), potato (Butler et al, 2015), FIGURE 4 | Strategy redesigning biosynthetic pathways for w-7 FAs production in common oil seeds.…”

Section: Crispr/case9 Gene Editing Technology In Designer Oil Productionmentioning

confidence: 99%

Metabolic Engineering a Model Oilseed Camelina sativa for the Sustainable Production of High-Value Designed Oils

Yuan

2020

Front. Plant Sci.

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Camelina sativa (L.) Crantz is an important Brassicaceae oil crop with a number of excellent agronomic traits including low water and fertilizer input, strong adaptation and resistance. Furthermore, its short life cycle and easy genetic transformation, combined with available data of genome and other "-omics" have enabled camelina as a model oil plant to study lipid metabolism regulation and genetic improvement. Particularly, camelina is capable of rapid metabolic engineering to synthesize and accumulate high levels of unusual fatty acids and modified oils in seeds, which are more stable and environmentally friendly. Such engineered camelina oils have been increasingly used as the super resource for edible oil, health-promoting food and medicine, biofuel oil and high-valued chemical production. In this review, we mainly highlight the latest advance in metabolic engineering towards the predictive manipulation of metabolism for commercial production of desirable bio-based products using camelina as an ideal platform. Moreover, we deeply analysis camelina seed metabolic engineering strategy and its promising achievements by describing the metabolic assembly of biosynthesis pathways for acetyl glycerides, hydroxylated fatty acids, medium-chain fatty acids, w-3 long-chain polyunsaturated fatty acids, palmitoleic acid (w-7) and other high-value oils. Future prospects are discussed, with a focus on the cutting-edge techniques in camelina such as genome editing application, fine directed manipulation of metabolism and future outlook for camelina industry development.

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Section: Crispr/case9 Gene Editing Technology In Designer Oil Productionmentioning

confidence: 99%

Metabolic Engineering a Model Oilseed Camelina sativa for the Sustainable Production of High-Value Designed Oils

Yuan

2020

Front. Plant Sci.

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“…Haploid cell lines such as HAP1 cells are an attractive model for genetic perturbation studies since the presence of a single allele implies enhanced knockout efficiency (Blomen et al 2015). Although we could show that knockout efficiency is only slightly decreased in diploid compared to haploid HAP1 cells, the effect is considered to be stronger in polyploid cancer cell lines where a full knockout requires out-of-frame editing of all alleles of a gene (Van Campenhout et al 2019) . HAP1 cells with specific gene knockouts have therefore been applied in many studies addressing various research questions (Davis et al 2015;Schick et al 2019;Lenk et al 2019;Smits et al 2019;Gerhards et al 2018;Baggen et al 2019) .…”

Section: Discussionmentioning

confidence: 67%

Pooled CRISPR screening at high sensitivity with an empirically designed sgRNA library

Henkel

Rauscher

Schmitt

et al. 2020

Preprint

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Given their broad utility in functionally annotating genomes, the experimental design of genome-scale CRISPR screens can vary greatly and criteria for optimal experimental implementation and library composition are still emerging. In this study, we report advantages of conducting viability screens in selected Cas9 single cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg)RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a new genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. In summary, we show how empirically designed libraries in combination with an optimised experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.

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“…Single guide RNAs (sgRNAs) targeting the human FKBP5 and PPARG genes were designed either using the Broad institute RNA guide design web tool (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) or obtained from Integrated DNA Technologies (IDT) and Thermo Fisher as pre-existing designs. All guide RNA designs were also evaluated by standard guide RNA design considerations before chosen for experiments 29 . The efficiency of three sgRNAs was assessed (see results under assessment of knockout efficiency section) for each gene after which the best performing guide was chosen for subsequent experiments.…”

Section: Crispr/cas9 Mediated Gene Knockout In Human Adipose Tissue-dmentioning

confidence: 99%

Proof-of-concept for CRISPR/Cas9 gene editing in human preadipocytes: Deletion of FKBP5 and PPARG and effects on adipocyte differentiation and metabolism

Kamble

Hetty

Vranic

et al. 2020

Sci Rep

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CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology. Adipose tissue is widely regarded as an endocrine organ that plays a central role in both obesity and insulin resistance 1. Dysregulation of adipose tissue transcriptional pathways may contribute to significant changes in energy balance, glucose and lipid metabolism, and adipokine secretion, which in turn can influence the whole-body metabolic homeostasis. Thus, identification of genes and functional assessment of their corresponding proteins involved in such pathways could help discover novel disease mechanisms that could be used for drug development. Different approaches such as pharmacological inhibition using receptor antagonists or neutralizing antibodies, as well as genetic manipulation using small interfering RNA-mediated knockdown 2 have been widely opted for studying the function of different gene products in human adipose cells. However, the specificity and stability of such approaches are critical factors and may sometimes limit their use. The recent advancements in clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology have given rise to a precise and highly efficient method for gene editing in cells, tissues and whole organisms 3. However, its application to human adipose tissue is scarce. To the best of our knowledge, only one study reported using this technique in primary human adipose cells, but the phenotypic results following a single nucleotide substitution in the fat mass and obesity-associated gene were equivocal 4. In another study, immortalized human brown preadipocytes were used to knockout genes using CRISPR/Cas9 5. Both of these studies have used an expression vector to deliver the CRISPR components into the cells. However, there are some potential complications associated with the use of plasmid DNA, as mentioned in the discussion.

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Guidelines for optimized gene knockout using CRISPR/Cas9

Cited by 37 publications

References 77 publications

Metabolic Engineering a Model Oilseed Camelina sativa for the Sustainable Production of High-Value Designed Oils

Metabolic Engineering a Model Oilseed Camelina sativa for the Sustainable Production of High-Value Designed Oils

Pooled CRISPR screening at high sensitivity with an empirically designed sgRNA library

Proof-of-concept for CRISPR/Cas9 gene editing in human preadipocytes: Deletion of FKBP5 and PPARG and effects on adipocyte differentiation and metabolism

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