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CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing [10] was the AAAS's choice for Breakthrough of the Year in 2015. [11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos. [12]
Targeted gene knockout using CRISPR/Cas9 requires the use of a delivery system to introduce the sgRNA and Cas9 into the cell. Although a number of different delivery systems are potentially available for CRISPR, [37] [38] genome-wide loss-of-function screens are predominantly carried out using third generation lentiviral vectors.
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and open up specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within living organisms.
Therefore, the GCN4-sfGFP-NLS-VP64 protein was developed to be interact with dCas SunTag system. The antibodies successfully bound to SunTag polypeptides and activated target CXCR4 gene in K562 cell lines. [11] Comparing with the dCas9-VP64 activation complex, they were able to increase the CXCR4 gene expression 5-25 times greater in K562 cell ...
CRISPR-based gene knockout is a powerful tool for understanding the genetic basis of disease and for developing new therapies. It is important to note that CRISPR-based gene knockout, like any genetic engineering technique, has the potential to produce unintended or harmful effects on the organism, so it should be used with caution.
CRISPR-associated endonuclease: Cas9 or other CRISPR-associated endonucleases such as Cpf1 must be introduced to cells that do not endogenously express them. Due to the large size of these genes, a two-vector system can be used to express the endonuclease separately from the sgRNA expression vector.
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