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C10184V7 Medical Science Question: Discuss about the Biological Function of CRISPR Technology.     Answer: Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR) are prokaryotic DNA segments with short and repetitive base sequences. (Cong et al. 2013). The actual biological function of CRISPR and Cas genes is seen in adaptive immunity of archaea and selected bacteria. With the help of these genes, the organisms are able to have an appropriate response to the genetic material and eliminate it consequently. There are three kinds of CRISPR mechanisms, among which the type II is mostly analyzed by researchers. In type II, the foreign DNA is suitably cut into smaller parts and entered into the CRISPR locus within a series of short repeats. These repeats are of 20 base pairs. The loci are then transcribed and transcripts are subsequently processed for the generation of small RNAs. These RNAs are used for guiding effector endonucleases targeting DNA on the basis of sequence complementarity. Cas9 protein, a kind of Cas protein, plays a significant role in CRISPR mechanisms. This Cas9 protein plays a role in gene silencing. It takes part in the processing of crRNA and also plays a role in the destruction of the DNA that is made the target. The function of the Cas9 in two steps is dependent on the action of two nuclease domains. One of these is the RuvC-like nuclease domain that is located at the amino terminus and the second is the HNH-like nuclease domain whose location is in the mid-region of the protein. For achieving the site-specific recognition and cleavage of the DNA, Cas9 has to form a complex with the crRNA as well as with the separate trans-activating crRNA. The trans-activating crRNA and the crRNA are partially complementary. For the maturation of crRNA from a primary transcript encoding, there is a requirement for the tracrRNA. The maturation leads to the encoding of multiple pre-crRNAs. The presence of Cas9 and RNase III is needed in this regard. At the time of the destruction of DNA that is made the target, the RuvC-like and HNH nuclease domains have to cut two of the DNA strands, as a result of which double-stranded breaks (DSBs) are generated at location where 20-nucleotide target sequence are present in linked crRNA transcript. The RuvC domain plays a role in cleaving the non-complimentary strand whereas the HNH domain function is to cleave the complementary strand. The double-stranded endonuclease activity exhibited by the Cas9 also needs the presence of a short conserved sequence, (2–5 nts) known as the protospacer-associated motif (PAM). It needs to follow immediately the 3´- of the crRNA complementary sequence. As a matter of fact, if PAM is absent then Cas9-RNA does not recognise the fully complementary sequences. The plainness of the type II CRISPR nuclease, as it requires only three components (Cas9, crRNA and trRNA) makes the system acquiescent to alteration for editing of genome (Hsu, Lander and Zhang, 2014).  The CRISPR method is being used for the introduction of single point mutations, insertions or deletions, in a certain gene that is targeted through the help of single gRNA (Guide-RNA). With the utilisation of gRNA-directed Cas9 nuclease, it is also possible to delete large genomic rearrangements, like translocations and inversions. The latest development is the application of a dCas9 version of the CRISPR/Cas9 system to protein domains that are made the target for regulation of transcription, epigenetic modification and visualisation of the specific genome loci. The CRISPR/Cas9 system needs a redesigning in the crRNA for changing target specificity. This is in contrast with genome editing tools, like zinc finger and TALENs, that needs redesigning of the protein-DNA interface (Zhang et al. 2015).   References Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A. and Zhang, F., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), pp.819-823. Hsu, P.D., Lander, E.S. and Zhang, F., 2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), pp.1262-1278. Peel, N. (2017). CRISPR gene editing: new chapter in cancer research or blot in the ethical copybook?. [online] Cancer Research UK – Science blog. Available at: [Accessed 19 Mar. 2017]. Zhang, Y., Yeo, W.S., Ng, K.H. and Yap, H.K., 2015. CRISPR/Cas9 for Genome Engineering: the Next Genomic Revolution. Journal of Biochemistry and Molecular Biology Research, 1(4), pp.112-117.

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