Over the past decades, progression in genetic element manipulation, and consequently, the treatment of diseases has been remarkable. It is worth noting that these genetic manipulations perform at different levels, including DNA and RNA. The earlier genomic editing techniques, including MN, ZFN, TALEN, performing their functions by creating double-stranded breaks (DSBs), and after breakage, the cell tries to repair the breakage through two systems, homologous recombination and non-homologous end joining. CRISPR/Cas technology has been discovered recently, and has become the most widely used genomeediting tool, mainly due to its capabilities and those added to this through the genetic engineering. In this study, we aimed to introduce a variety of CRISPR classes in the elementary parts, and then the modified CRISPR systems developed to increase the efficiency and specificity of the system and provide acceptable results will be introduced. In this study, for three months in the fall and winter, Pubmed and Web of science sites searched for keywords such as CRISPR, Types of CRISPR, gRNA, Cas9, and CRISPR-Cas9 nickase that eventually resulted in about four hundred Sixty-one articles, and some of these articles after closer study, reviewed in this article. Genetic engineering techniques have successfully transformed this system into the most efficient genome editing tool in recent years. Researchers are working on a system to treat various diseases by resolving problems such as high specificity, cutting off non-target sites, how to move to a cell, and setting up a proper repair system.

Targeted genome editing by RNA-guided nucleases is a high throughput and facile approach which not only provides modifications, gene editing and functional studies for researchers, but also develops their knowledge about molecular basis of diseases and stablishes novel targeted therapies. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). Indeed CRISPR/Cas9 system is bacterial defensive system against viral invasions. Cas9 protein can be targeted to DNA sequences by accompanying single-strand RNA guide which base-pairs directly with target DNA to introduce modifications. Improvements are urgently needed for various aspects of system, including delivery and control over the outcome of the repair process. CRISPR/Cas9 holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites the field of Cas9 specificity is rapidly progressing with marked improvements in guide RNA selection, novel enzymes and off-target detection methods. In this review we discuss history and different aspects of CRISPR/Cas9 including advances in specificity strategies for genome engineering, system delivery and control optimization on repair processes along with its implication for the future of biological researches and gene therapy.

Plant diseases caused by viruses are a serious threat to the world's agricultural production, and antivirus engineering, initiated by molecular biotechnology, is an effective strategy for preventing and controlling plant-borne viruses. Recent advances in targeted DNA or RNA editing with a short palindromic replication system or CRISPR-related are attractive tools that can be used to protect plants. In this review, the development of CRISPR/Cas systems is surved and their programs in controlling various plant viruses by targeting viral sequences or susceptibility genes in the host are mentioned. In addition, it introduces some potentially recessive resistance genes that can be used to improve plants against viruses, and emphasizes the importance of recessive gene-based antiviral modification for producing plants free of virus and growth deficiency. In addition, the challenges and opportunities of using CRISPR/Cas techniques in the prevention and control of plant viruses are discussed.

Background: Engineered molecular scissors invention beside their ability to conduct site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes. These technologies could improve researchers’ understanding of the molecular underpinnings of disease states and facilitate novel medical genetic therapeutic applications. The CRISPR/Cas9 system’ s simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. It is expected that in the near future researchers will refinement this technology in various aspects of the CRISPR/Cas9 system, including the system’ s precision, delivery and control over the outcome of the on-targrt activity during repair process. Here, we discuss the CRISPR/Cas9 history, type of CRISPR systems, the status of this technology in the field of genome editing and its use in new treatments for diseases such as genetics, cancer, etc.

Background and Objectives: The genome of cell lines is nowadays edited to create disease models and treat them. Of course, the size of the cas9 gene has caused problems like the low efficiency of the CRISPR system. To solve this problem, Cas9 expressing cell lines have been generated in which CRISPR RNA should only be transfected to the cell. Materials and Methods: This article is experimental. PGK-PURO / CMV (PPC) fragment was amplified with PCR from pAAVS1-puro-DNR vector and cloned in pTG19-T vector. The PPC fragment from this vector was removed by KpnI and EcoRI enzymes. Also the pCas-Guide-AAVS1 vector was subjected to the same enzymatic cutting and its attachment to the PPC fragment resulted in the production of the pPPC-Cas vector. After optimizing the electroporation conditions, the pPPC-Cas vector was electroporated into K562 cells and puromycin-resistant cells were selected and Cas9 expression level was evaluated by Real-time PCR. Results: A PCR fragment of 2514 bp was amplified. The vector pPPC-Cas was cloned in two steps. puromycin-resistant transfected cells were selected. Clonal selection was carried out and three colonies with high, medium and low expression level of Cas9 were isolated. Conclusions: The Cas9-expressing K562 cells derived in this study can be applied both for functional genomic researches and design cellular models of human diseases in future.

Rice (Oryza sativa L. ) is the major food source for the most of people of the world. In the last few decades, the classical, mutational and molecular breeding approaches have caused enormous increase in rice productivity with the development of novel rice varieties. Stagnation in rice yield has been reported in recent decade because of emergence of pests and phyto pathogens, climate change, and other environmental issues. The CRISPR/Cas9 system has all genome editing capabilities, e. g., knock-in, knockout, knockdown, and expression activation. Researchers have been successful in editing genes controlling traits such as number of panicles per plant, number of seeds per panicle, grain weight, rice maturity, plant hormone synthesizing genes, amylose content, gelatinization temperature, grain length and width, as well as resistance to abiotic and biotic stresses. The shift of research toward the utilization of CRISPR/Cas9 systems for targeted mutagenesis could be a promising approach for overcoming the barriers to breeding of improved rice. In this review, the progress in rice by employing the CRISPR/Cas9 editing system and its famous applications have been discussed. It is seemed that the CRISPR/Cas9 and associated genome editing tools have taken in a revolutionary change in rice improvement which is very important for meeting the demands and ensuring the requirement of rice for future generations.