Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells

Zheng, Qiuling; Cai, Xiaohong; Tan, Meng How; Schaffert, Steven; Arnold, Carrie; Gong, Xue; Chen, Changzheng; Huang, Shenglin

doi:10.2144/000114196

Cited by 132 publications

(79 citation statements)

References 23 publications

(13 reference statements)

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“…Use of two gRNAs coupled with Cas9 can efficiently create DNA deletions of up to 10 kb in the presence of a linear homologous repair donor [108]. Interrogation of gene function on a genome-wide scale can be facilitated by large scale oligonucleotide synthesis of guide sequences [109].…”

Section: Functional Genomics By Crispr-cas9mentioning

confidence: 99%

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Shen¹,

McHale²,

Smith³

et al. 2015

Mutation Research/Reviews in Mutation Research

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Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells, have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide unprecedented mechanistic insight in the field of modern toxicology.

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Section: Functional Genomics By Crispr-cas9mentioning

confidence: 99%

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Shen¹,

McHale²,

Smith³

et al. 2015

Mutation Research/Reviews in Mutation Research

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show abstract

“…2A). Targeting a cellular gene with two gRNA/Cas9 complexes will result, in many cases, in a specific gene fragment deletion (10). To mutate the cGAS and STING genes, two gRNA sequences per gene were designed to delete a 32-bp (cGAS) and 149-bp (STING) fragment just downstream of the translation initiation site (Fig.…”

mentioning

confidence: 99%

Knockout of cGAS and STING Rescues Virus Infection of Plasmid DNA-Transfected Cells

Langereis

Rabouw

Holwerda

et al. 2015

J Virol

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It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here, we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, we generated cGAS/STING-knockout cells and show that these cells can be infected after plasmid DNA transfection as efficiently as nontransfected cells. Mammalian cells express various receptors to sense invading pathogens. Specialized receptors that survey the intracellular milieu are the NOD-like receptors (NLRs), the RIG-I-like receptors (RLRs), and numerous receptors that detect cytosolic DNA (1). Recently, a ubiquitously expressed DNA sensor, cyclic GMP-AMP synthase (cGAS), has been identified (2, 3). Upon binding of cytosolic DNA, like herring testis DNA and poly(dA: dT), cGAS produces cyclic GMP-AMP signaling molecules that subsequently activate stimulator of interferon genes (STING [4,5]). STING then activates a cascade that results in the synthesis and secretion of type I interferons (interferon alpha/beta [IFN-␣/ ␤]), ultimately leading to the induction of an antiviral state of the cells.Plasmid DNA transfection is often used as a transient gene delivery system to overexpress proteins (e.g., dominant negative or green fluorescent protein [GFP] fusion proteins). Unfortunately, in many cases the transfection procedure blocks virus infection. Although it is conceivable that transfected plasmid DNA activates the cGAS/STING pathway, which might lead to an antiviral state of the cells, this has not been properly investigated.To study the plasmid DNA-mediated repression of virus infection, we transfected red fluorescent protein (RFP)-encoding plasmid DNA into HeLa-R19 and BGM cells using Fugene 6 (similar experiments, with comparable results, have been performed using Lipofectamine 2000 and JetPRIME [data not shown]). Twentyfour hours posttransfection, the cells were infected with the coxsackievirus B3-enhanced GFP (CVB3-EGFP) and mengovirus-EGFP (mengo-EGFP) reporter viruses, and infection efficiency was monitored using flow cytometry analysis. We observed a marked decrease in CVB3-EGFP infection efficiency in plasmid DNA-transfected HeLa-R19 cells (from 70% to 31%) and BGM cells (from 43% to 11%) compared to mock-treated cells (Fig. 1A). A comparable decrease in infection efficiency was also observed for mengo-EGFP (Fig. 1B). It is important to note that virus infection was hampered in both RFP-positive and RFP-negative cells. This suggests that plasmid DNA transfection induces the secretion of a soluble factor that restricts virus infection, possibly cGAS/STING-induced IFN-␣/␤. To investigate this, we performed transfection/infection experiments in HEK293T and Vero-E6 cells, which are known to be deficient in cGAS and STING signaling (3, 6) and the production of IFN-␣/␤ (7), respectively. In these cells, plasmid DNA transfection...

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“…Four out of six (M2_9, M2_10, M3_10 and M3_11) showed the expected 37 nt ‘clean’ deletion without any additional mutations. Precise deletions, such as this, using 2 sgRNAs have previously been generated with high efficiency [37, 42], and allow a large degree of control over the mutation. Two of the sub-clones (M3_9 and M2_12) showed one allele with the expected 37 nt deletion and the other with an additional deletion at the 2nd sgRNA cut site.…”

Section: Resultsmentioning

confidence: 99%

“…Precise deletions larger than a gene using CRISPR-Cas and two sgRNAs have been previously demonstrated [42]. …”

Section: Resultsmentioning

confidence: 99%

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

et al. 2016

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BackgroundCRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.ResultsA single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.ConclusionsCRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0148-0) contains supplementary material, which is available to authorized users.

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Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells

Cited by 132 publications

References 23 publications

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9

Knockout of cGAS and STING Rescues Virus Infection of Plasmid DNA-Transfected Cells

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

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