Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences

Bae, Taegeun; Kim, Hanseop; Kim, Jeong Hee; Kim, Yong Jun; Lee, Seung Hwan; Ham, Byung Joo; Hur, Junho K.

doi:10.3390/molecules25010052

Cited by 6 publications

(4 citation statements)

References 24 publications

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“…The lack of evidence of the direct effect of DNA methylation on gene expression is an important limitation of these studies as the methylation status of a particular CpG site could affect the expression and function of several other DNA elements in addition to the promoter of the closest gene [ 14 ]. The next step in this research field would be a gene-specific modification of the DNA methylation status with a CRISPR/Cas9 related system to validate its effect on gene expression [ 104 , 105 ]. Furthermore, modulation of DNA methylation at specific CpG sites or a single-base mutation within a CpG (associated with a methylation quantitative trait loci) could be performed using this technology to assess the effects on gene expression.…”

Section: Dna Methylationmentioning

confidence: 99%

The role of epigenetic mechanisms in the regulation of gene expression in the cyclical endometrium

et al. 2021

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Background The human endometrium is a highly dynamic tissue whose function is mainly regulated by the ovarian steroid hormones estradiol and progesterone. The serum levels of these and other hormones are associated with three specific phases that compose the endometrial cycle: menstrual, proliferative, and secretory. Throughout this cycle, the endometrium exhibits different transcriptional networks according to the genes expressed in each phase. Epigenetic mechanisms are crucial in the fine-tuning of gene expression to generate such transcriptional networks. The present review aims to provide an overview of current research focused on the epigenetic mechanisms that regulate gene expression in the cyclical endometrium and discuss the technical and clinical perspectives regarding this topic. Main body The main epigenetic mechanisms reported are DNA methylation, histone post-translational modifications, and non-coding RNAs. These epigenetic mechanisms induce the expression of genes associated with transcriptional regulation, endometrial epithelial growth, angiogenesis, and stromal cell proliferation during the proliferative phase. During the secretory phase, epigenetic mechanisms promote the expression of genes associated with hormone response, insulin signaling, decidualization, and embryo implantation. Furthermore, the global content of specific epigenetic modifications and the gene expression of non-coding RNAs and epigenetic modifiers vary according to the menstrual cycle phase. In vitro and cell type-specific studies have demonstrated that epithelial and stromal cells undergo particular epigenetic changes that modulate their transcriptional networks to accomplish their function during decidualization and implantation. Conclusion and perspectives Epigenetic mechanisms are emerging as key players in regulating transcriptional networks associated with key processes and functions of the cyclical endometrium. Further studies using next-generation sequencing and single-cell technology are warranted to explore the role of other epigenetic mechanisms in each cell type that composes the endometrium throughout the menstrual cycle. The application of this knowledge will definitively provide essential information to understand the pathological mechanisms of endometrial diseases, such as endometriosis and endometrial cancer, and to identify potential therapeutic targets and improve women’s health.

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Section: Dna Methylationmentioning

confidence: 99%

The role of epigenetic mechanisms in the regulation of gene expression in the cyclical endometrium

et al. 2021

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“…It is the third generation of enzyme-related gene editing tool, right after zinc-finger nucleases (ZFN) and transcription activator-like effectors nucleases (TALENS) 3 . The CRISPR/Cas9 system was initially discovered as the "immune system" for bacteria and archaea, which uses this strategy to fight against viruses that inject DNA into them [6][7][8][9] . The system has a prominent aspect as it only requires designing a singleguided RNA (sgRNA), which usually consists of twenty base pairs, that is capable of leading the Cas9 enzyme to bind to the site where the DNA sequence needs to be modified [10][11][12][13][14] .…”

Section: Introductionmentioning

confidence: 99%

Prediction of off-target effects of the CRISPR/Cas9 system for design of sgRNA

Guo

Zhen

2020

E3S Web Conf.

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CRISPR/Cas9 genome editing technology is the frontier of life science research. They have been used to cure human genetic diseases, achieve cell personalized treatment, develop new drugs, and improve the genetic characteristics of crops and other fields. This system relies on the enzyme Cas9 cutting target DNA (on target) under the guidance of sgRNA, but it can also cut non-target sites, which results in offtarget effects, thus causing uncontrollable mutations. The risk of off-target effect in CRISPR technology is the main limiting factor that affects the widespread application of CRISPR technology. How to evaluate and reduce the off-target effect is the urgent problem to be solved. In this work, we build up a model that can predict the score of being off-target. Through comparison with the complete genome of the target and precise mathematics that calculate the potential risk of being off-target, we optimize the sgRNA, which is capable of reducing the off-target effect. The result has proven that we can efficiently and quickly identify and screen the best editing target sites with our model. The CRISPR/Cas9 system, not even being perfected yet, has already demonstrated its potential in the field of genome editing. Hopefully through our model, the CRISPR/Cas9 system can quickly apply to more branches in life science and cure those diseases that have been previously incurable.

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“…CRISPR-associated protein 9 (CRISPR/Cas9), one of the most widely used genome editing tools, consists of two components: single guide RNA (sgRNA) and Cas9 protein . SgRNA can specifically recognize the target via hybridizing with the target sequence and form dsDNA, which can be recognized and cleaved by Cas9. − It should be noted in particular that, with the aid of the nuclease, sgRNA can specifically recognize the target with single-base mismatch within 10 bases close to the PAM. , …”

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confidence: 99%

“…19−21 It should be noted in particular that, with the aid of the nuclease, sgRNA can specifically recognize the target with single-base mismatch within 10 bases close to the PAM. 22,23 Electrochemical detection platforms have been widely applied in the detection of biomarkers owing to their advantages of low cost and quick speed. The multichannel series piezoelectric quartz crystal (MSPQC) sensor has the characteristics of sensitive, rapid, low-cost, and easy-toimplement point-of-care testing (POCT) detection.…”

mentioning

confidence: 99%

Development of an MSPQC Nucleic Acid Sensor Based on CRISPR/Cas9 for the Detection of Mycobacterium tuberculosis

Huang

Liu

et al. 2022

Anal. Chem.

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Accurate and rapid detection of nucleic acid plays a vital role in the clinical treatment of tuberculosis caused by Mycobacterium tuberculosis (M.TB). However, false-negative and false-positive results caused by base mismatches could affect the detection accuracy. Inspired by the unique property of CRISPR/Cas9, we proposed a new MSPQC M.TB sensor based on the CRISPR/Cas9 system, which can distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. In the proposed sensor, single-stranded DNA on Au interdigital electrodes was used as a capture probe for the target and an initiator for hybridization chain reaction (HCR). HCR was used to generate long double-stranded DNA (dsDNA), which could span the Au interdigital electrodes. CRISPR/Cas9 was used as recognition components to recognize capture/target dsDNA. When the target existed, the capture probe hybridized with the target to form dsDNA, which could be recognized and cut by CRISPR/Cas9. Thus, the DNA connection between electrodes was cut off and resulted in the MSPQC response. When no target existed, the capture probe remained single-stranded and could not be recognized and cut by CRISPR/Cas9. Therefore, DNA connection between electrodes was reserved. Moreover, silver staining technology was utilized to improve the sensitivity of detection. M.TB was detected by the proposed sensor using specific sequence fragments of 16S rRNA of M.TB as the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.

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Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences

Cited by 6 publications

References 24 publications

The role of epigenetic mechanisms in the regulation of gene expression in the cyclical endometrium

The role of epigenetic mechanisms in the regulation of gene expression in the cyclical endometrium

Prediction of off-target effects of the CRISPR/Cas9 system for design of sgRNA

Development of an MSPQC Nucleic Acid Sensor Based on CRISPR/Cas9 for the Detection of Mycobacterium tuberculosis

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