A CRISPR view of development

Harrison, Melissa M.; Jenkins, Brian V.; O’Connor-Giles, Kate M.; Wildonger, Jill

doi:10.1101/gad.248252.114

Cited by 205 publications

(196 citation statements)

References 147 publications

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“…Among those techniques are transcription activator-like effector nucleases (TALENs) that use a pair of artificial DNA-binding domains fused to the catalytic domain of restriction endonuclease FokI which causes a double-strand break (DSB) at the targeted genomic locus stimulating DNA repair (6,7). Yet, TALEN is labor-intensive and works with low efficiency (8).…”

Section: Introductionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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Abstract. Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl --1, HLF-Axl --2, SNU449-Axl --1, SNU449-Axl --2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl -clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region.However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl --1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl --1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

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Section: Introductionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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“…The second mechanism, homologous recombination (HR), is based on a template homologous to the sequence surrounding the DSB. The template is present in the chromosome in case of a naturally occurring HR; however, if an external template is delivered, the HR can be used to make custom changes to the genome including insertions of an exogenous DNA sequence (Harrison et al, 2014;Belhaj et al, 2015;Bortesi and Fischer, 2015). Due to the provided template, the change made by HR is usually exact (Belhaj et al, 2015); however, compared to NHEJ, it occurs…”

Section: Site-directed Nucleases: Methods and Applicationsmentioning

confidence: 99%

“…Non-homologous end joining (NHEJ) is the main pathway the cells use to repair DSBs and involves the exposed DNA ends being directly reconnected. Since NHEJ is error prone, the repair is often associated with insertions or deletions (together called indels) of one or more nucleotides (Harrison et al, 2014;Rinaldo and Ayliffe, 2015). If the DSB creates overhangs (i.e.…”

Section: Site-directed Nucleases: Methods and Applicationsmentioning

confidence: 99%

“…The ZFNs are composed of two independent regions: a recognition domain of zinc fingers each identifying a nucleotide triplet of the target DNA sequence and a non-specific nuclease, called FokI, creating the DSB (Harrison et al, 2014;Rinaldo and Ayliffe, 2015). Since the nuclease needs to dimerize in order to be active, the ZFNs are used in pairs.…”

Section: Zinc Finger Nucleasesmentioning

confidence: 99%

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Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

2017

Current Issues in Molecular Biology

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“…Some of the designer nucleases are clustered as regularly interspaced short palindromic repeats/Cas9, 116 transcription activator-like effector nucleases, and zinc finger nucleases. 117 These technologies can be used to generate gene knockouts or gene knockins when applied alone or in combination with donor DNA templates, respectively.…”

Section: Future Perspectivementioning

confidence: 99%

Genomic and Epigenomic Alterations in Cancer

Chakravarthi

Nepal

Varambally

2016

The American Journal of Pathology

154

101

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Multiple genetic and epigenetic events characterize tumor progression and define the identity of the tumors. Advances in high-throughput technologies, like gene expression profiling, next-generation sequencing, proteomics, and metabolomics, have enabled detailed molecular characterization of various tumors. The integration and analyses of these high-throughput data have unraveled many novel molecular aberrations and network alterations in tumors. These molecular alterations include multiple cancer-driving mutations, gene fusions, amplification, deletion, and post-translational modifications, among others. Many of these genomic events are being used in cancer diagnosis, whereas others are therapeutically targeted with small-molecule inhibitors. Multiple genes/enzymes that play a role in DNA and histone modifications are also altered in various cancers, changing the epigenomic landscape during cancer initiation and progression. Apart from protein-coding genes, studies are uncovering the critical regulatory roles played by noncoding RNAs and noncoding regions of the genome during cancer progression. Many of these genomic and epigenetic events function in tandem to drive tumor development and metastasis. Concurrent advances in genome-modulating technologies, like gene silencing and genome editing, are providing ability to understand in detail the process of cancer initiation, progression, and signaling as well as opening up avenues for therapeutic targeting. In this review, we discuss some of the recent advances in cancer genomic and epigenomic research.

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A CRISPR view of development

Cited by 205 publications

References 147 publications

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

Genomic and Epigenomic Alterations in Cancer

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