Advances in Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated system (CRISPR/Cas9) has dramatically reshaped our ability to edit genomes. The scientific community is using CRISPR/Cas9 for various biotechnological and medical purposes. One of its most important uses is developing potential therapeutic strategies against diseases. CRISPR/Cas9 based approaches have been increasingly applied to the treatment of human diseases like cancer, genetic, immunological and neurological disorders and viral diseases. These strategies using CRISPR/Cas9 are not only therapy oriented but can also be used for disease modeling as well, which in turn can lead to the improved understanding of mechanisms of various infectious and genetic diseases. In addition, CRISPR/Cas9 system can also be used as programmable antibiotics to kill the bacteria sequence specifically and therefore can bypass multidrug resistance. Furthermore, CRISPR/Cas9 based gene drive may also hold the potential to limit the spread of vector borne diseases. This bacterial and archaeal adaptive immune system might be a therapeutic answer to previous incurable diseases, of course rigorous testing is required to corroborate these claims. In this review, we provide an insight about the recent developments using CRISPR/Cas9 against various diseases with respect to disease modeling and treatment, and what future perspectives should be noted while using this technology.
Over the last decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR/Cas9 system has been used by biologists in various fields. In plant biology, the technology is being utilised to manufacture transgenic plants resistant to different diseases. In Pakistan, the Cotton leaf curl virus (CLCuV) (a begomovirus) affects cotton plants causing significant loss to the economy of this agriculturally based country. In the present study, we use the CRISPR/Cas9 system in Nicotiana benthamiana Domin (a model plant) to develop resistance against CLCuV. An interesting facet of the study was the comparison of two constructs (pHSE401 and pKSE401) with regards to their efficacy in the virus inhibition. The pKSE401 vector contained a Cas9 nuclease and two guide RNAs (gRNAs), one targeting the Replication associated protein (Rep) gene and the other targeted the βC1 gene of the Betasatellite. The vector pHSE401 had only one sgRNA that targeted the (Rep) gene. Both genes that are intended to be targeted play important roles in the replication of CLCuV. Plants infiltrated with pKSE401 exhibited a delay in the development of the symptoms of the disease and showed lower virus titres. Our proposed multiplexing approach gave efficient results of the resistance in the model plants, and the results in this communication may be extended to the CRISPR/Cas9 based editing of cotton plants.
Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Mosaic Virus (ToMV) are the major diseases/viruses. A set of 40 accessions (including indigenous Pakistani lines and exotic germplasm from Europe, the United States, and Asia) were evaluated for their resistance/infection response to ToMV with artificial inoculation under greenhouse conditions. Infection response was quantified through disease scoring and DAS-ELISA test (for ToMV). A subset of 24 lines, was further screened for TYLCV using disease scoring and TAS-ELISA. The tested lines showed significant variability for resistance to ToMV. Only one accession (Acc-17878) was resistant to the ToMV whereas seven accessions i.e. Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362 expressed resistance to TYLCV. Correlation between phenotypic evaluation was confirmed by the ELISA results in both diseases, although both tools complemented to assess the viral infection status. In future, tomato breeding programs must consider breeding for ToMV and TYLCV resistance (using identified germplasm in our study) so as to deliver virus resistant tomato varieties.
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