Nuclease Architecture Efficient Genome Editing

Question: Discuss about the Nuclease Architecture for Efficient Genome Editing. Answer: Introduction: The homologous recombination method could be used to correct the point mutation in sickle cell anemia. The iPSCs of the sickle cell anemia patient having the mutation at both the globin alleles are derived and a mechanism of plasmid mediated gene targeting having a gene cassette of loxP flanked drug resistant to target and select the rare clones. The homologous recombination is done by zinc finger nucleases at the beta locus having mutated sickle cell allele. This method is useful as it provides the correction of one s allele present at the locus. The second mutated allele is corrected by excision mediated by Cre recombinase of the flanked loxP gene cassette that helped to express the wild type allele by 40% when they differentiated in erythrocytes (Zou et al. 2011). The non homologous end joining corrects the point mutation in sickle cell anemia. The TALENs enable genetic modifications by stimulating non-homologous end joining and inducing the error prone method at the specified genomic locations. The transcription activator-like effector nucleases are important in inducing therapeutic site specific nuclease technologies that stimulate the non homologous end joining at targeted beta globin gene in the sickle cell anemia patient. The TALENs fuse the nonspecific DNA cleaving nuclease to a DNA binding domain that could be easily engineered to target any kind of sequence like the beta globin gene sequence in case of sickle cell anemia. It is a potential tool in the therapeutic strategy in correcting gene4tic diseases caused due to point mutation (Joung and Sander 2013). The non homologous end joining that involves the high capacity to repair and is active throughout cell cycle. It does not require a homologue or sister chromatid for the extensive synthesis of DNA. The non homologous end joining repairs the breaks in minutes and it provides higher magnitude than the homologous direct repair. The NHEJ is the means to repair the cas9 induced breaks. The NHEJ has a vast machinery and tool to correct the repair caused due to double stranded breaks. It consists of nucleases, polymerases and structure specific enzymes and it ensures better ligation than homologous direct repair mechanism. The processing of the DNA ends tends to be more processing where the mutations occur. The cas9 induced single stranded breaks are repaired by the targeted single stranded break the strategy of double nickase also reduces the mutations and breaks at the targets that are off sites. The knock in strategy of the exogenous DNA at a selected single gene locus mediates better ef ficiency of the joining of the plasmids and genome. The DNA integration is highly efficient in the CRISPR/cas9 induced double stranded breaks and efficient in genome editing in the somatic and human embryonic stem cells. References Joung, J.K. and Sander, J.D., 2013. TALENs: a widely applicable technology for targeted genome editing. Nature reviews Molecular cell biology, 14(1), pp.49-55. Miller, J.C., Tan, S., Qiao, G., Barlow, K.A., Wang, J., Xia, D.F., Meng, X., Paschon, D.E., Leung, E., Hinkley, S.J. and Dulay, G.P., 2011. A TALE nuclease architecture for efficient genome editing.Nature biotechnology,29(2), pp.143-148. Zou, J., Mali, P., Huang, X., Dowey, S.N. and Cheng, L., 2011. Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood, 118(17), pp.4599-4608.