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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.
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]
A gene "knockout" model for TBX5 by CRISPR/Cas9 genome editing has been created. [22] This homozygous TBX5 knockout human embryonic stem cell line, called TBX5-KO maintained stem cell-like morphology, pluripotency markers, normal karyotype, and could differentiate into all three germ layers in vivo.
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.
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.
The CRISPR-CAS9 system has the ability to either upregulate or downregulate genes. The dCas9 proteins are a component of the CRISPR-CAS9 system and these proteins can repress certain areas of a plant gene. This happens when dCAS9 binds to repressor domains, and in the case of the plants, deactivation of a regulatory gene such as AtCSTF64, does ...
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