CRISPR/Cas system: A game changing genome editing technology, to treat human genetic diseases

Hussain, Wajid; Mahmood, Tariq; Hussain, Jawad; Ali, Niyaz; Shah, Tariq; Qayyum, Sumaira; Khan, Ibrar

doi:10.1016/j.gene.2018.10.072

Cited by 37 publications

(20 citation statements)

References 35 publications

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“…The CRISPR gene sequence was composed of Cas protein gene, leader sequence and CRISPR locus, of which the nuclease was Cas9 endonuclease, and DNA double-strand cutting was performed through identification of PAM. This technology recognizes DNA sequences with PAM sequences through sgRNA, and brings Cas9 nuclease to specific targets on the genome to complete the cleavage of DNA sequences at specific gene sites ( 23 ). This technology is simple and fast to operate, with low associated costs and higher efficiency compared with traditional gene editing technologies ( 9 ).…”

Section: Discussionmentioning

confidence: 99%

Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

Cao

Xiao

et al. 2020

Oncol Lett

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Human epidermal growth factor receptor 2 (HER-2) + breast cancer has a high recurrence rate and a poor prognosis, with drug resistance contributing to disease progression. The present study aimed to establish a SKBR3 cell line with type II insulin-like growth factor receptor (IGR-IIR) gene site integration using the CRISPR/Cas9 system, and to provide a cell model for exploring the mechanism responsible for the effect of IGF-IIR on trastuzumab resistance in HER-2 + breast cancer cells. In the present study, six single guide (sg)RNA pairs according to the adeno-associated virus integration site 1 (AAVS1) gene sequence were designed and synthesized, and the Universal CRISPR Activity assay CRISPR/Cas9 rapid construction and activity detection kit was used to connect the annealed oligo with the pCS vector. The sgRNA with the highest efficiency was selected to construct a Cas9/sgRNA expression vector using Asi SI + Bst z17I restriction enzymes to cut IGF-IIR. The fragment was ligated into an human AAVS1-KI vector to construct the IGF-IIR targeting vector. The Cas9/sgRNA and IGF-IIR targeting vectors were electroporated into SKBR3 cells, screened using puromycin and identified via PCR, and the mixed cloned cells generated via IGF-IIR gene targeted integration were obtained. The semi-solid and limited dilution methods were used for monoclonal cell preparation, and the results revealed that a Cas9/sgRNA vector that targeted the AAVS1 was successfully constructed. sgRNA activity detection demonstrated that sgRNA2 had the highest efficiency, while enzyme digestion and sequencing confirmed that the IGF-IIR target vector was successfully constructed. The optimum conditions for electrotransfection were 1,200 V, 20 ms and 2 pulses, and the optimal screening concentration of puromycin was 0.5 µg/ml. Using these conditions, the IGF-IIR targeting vector and pCS-sgRNA2 plasmid were successfully transfected into SKBR3 cells, and PCR identification and sequencing verified the correct genotype of mixed clone fragments. The monoclonal cells proliferate slowly and gradually underwent apoptosis. Overall, the present study successfully obtained a mixed clone cell line with site-specific integration of the IGF-IIR gene at the AAVS1.

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Section: Discussionmentioning

confidence: 99%

Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

Cao

Xiao

et al. 2020

Oncol Lett

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“…The last four decades have seen the rise of nucleic acids as therapeutics [ 1 ], primarily in the form of antisense oligonucleotides (ASOs) [ 2 ], small interfering RNAs (siRNAs) [ 3 ], aptamers [ 4 – 6 ], microRNAs [ 7 ], mRNAs [ 8 ], and gene-editing guides [ 9 ]. Nucleic acids afford completely new modalities for therapeutic intervention (e.g., direct protein expression or specific and programmable modulation of gene expression) that cannot be easily accessed by other biologics or small molecule drugs.…”

Section: Nucleic Acids: Natural and Expanded Functionsmentioning

confidence: 99%

Modified nucleic acids: replication, evolution, and next-generation therapeutics

2020

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Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs.

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“…Moreover, the memory from the antigen is stored as spacer within CRISPR 24 This physiological role of Cas9/CRISPR as explained above had recently been extensively utilized for multiple clinical conditions 25, 26, 27. At the time of writing this review, the news broke about Lulu and Nana being claimed to be the first genetically modified babies, where the human genome was edited to create resistance against HIV infection 28 .…”

Section: Main Textmentioning

confidence: 99%

Genome-Editing Technologies: Concept, Pros, and Cons of Various Genome-Editing Techniques and Bioethical Concerns for Clinical Application

Khan

2019

Molecular Therapy - Nucleic Acids

173

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The traditional healthcare system is at the doorstep for entering into the arena of molecular medicine. The enormous knowledge and ongoing research have now been able to demonstrate methodologies that can alter DNA coding. The techniques used to edit or change the genome evolved from the earlier attempts like nuclease technologies, homing endonucleases, and certain chemical methods. Molecular techniques like meganuclease, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) initially emerged as genome-editing technologies. These initial technologies suffer from lower specificity due to their off-targets side effects. Moreover, from biotechnology's perspective, the main obstacle was to develop simple but effective delivery methods for host cell entry. Later, small RNAs, including microRNA (miRNA) and small interfering RNA (siRNA), have been widely adopted in the research laboratories to replace lab animals and cell lines. The latest discovery of CRISPR/Cas9 technology seems more encouraging by providing better efficiency, feasibility, and multi-role clinical application. This later biotechnology seem to take genomeengineering techniques to the next level of molecular engineering. This review generally discusses the various gene-editing technologies in terms of the mechanisms of action, advantages, and side effects.

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CRISPR/Cas system: A game changing genome editing technology, to treat human genetic diseases

Cited by 37 publications

References 35 publications

Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

Modified nucleic acids: replication, evolution, and next-generation therapeutics

Genome-Editing Technologies: Concept, Pros, and Cons of Various Genome-Editing Techniques and Bioethical Concerns for Clinical Application

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CRISPR/Cas system: A game changing genome editing technology, to treat human genetic diseases

Cited by 37 publications

References 35 publications

Establishment of a type&nbsp;II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

Establishment of a type&nbsp;II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

Modified nucleic acids: replication, evolution, and next-generation therapeutics

Genome-Editing Technologies: Concept, Pros, and Cons of Various Genome-Editing Techniques and Bioethical Concerns for Clinical Application

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Product

Resources

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Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9