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CRISPR-Based Genome Engineering in Human Stem Cells
Published in Deepak A. Lamba, Patient-Specific Stem Cells, 2017
Thelma Garcia, Deepak A. Lamba
For the most part, CRISPR–Cas9 systems provide DNA-encoded, RNA-mediated, DNA- or RNA-targeting sequence-specific targeting. Cas9 is the signature protein for Type II CRISPR–Cas9 systems. A ribonucleoprotein complex consisting of a CRISPR RNA (crRNA) in combination with a trans-activating crRNA and a Cas9 nuclease targets complementary DNA flanked by a protospacer adjacent motif (PAM) to obtain desired gene editing. There are three different variants of Cas9 nuclease: double nick, single nick, and mutant nick.
Site-specific integration of light chain and heavy chain genes of antibody into CHO-K1 stable hot spot and detection of antibody and fusion protein expression level
Published in Preparative Biochemistry and Biotechnology, 2019
Songtao Zhou, Yun Chen, Xiaohai Gong, Jian Jin, Huazhong Li
Site-specific recombinase based technology such as Cre/loxP system and Flp/FRT system[9–11] requires prior insertion of recombination site into the hot spot to achieve knock-in of target gene; however, the entire operation process is very complicated.[12,13] The Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) RNA-guided nucleases system or CRISPR/Cas system is readily available for fast and accurate genome editing within multiple species’ genomes.[14,15] The active cleavage-complex of CRISPR/Cas9 system is composed of CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA) and Cas9 nuclease. These elements can induce site-specific DNA double-strand breaks (DSBs). There are two major DNA repairing pathways following DSBs: nonhomologous end-joining (NHEJ) and homology-directed repair (HDR).[6,16] When provided with a donor DNA that carries homologous arms to both sides of the DSB, genomic site can be repaired precisely by HDR allowing the integration of genes of interest (GOI) between homology regions.[17] The targeting rate of CRISPR/Cas9-mediated HDR was reported to be ∼10% based on previous literature.[6] Here, the CRISPR/Cas9-based method was adopted to achieve site-specific integration of target gene.