CRISPR-Cas9 SYSTEM AS A GENOMIC TOOL FOR MANIPULATING AND TARGETING OF GENOMES
CRISPR-Cas9 endonuclease system is becoming a significant tool in genome editing processes. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) provides adaptive immunity to the bacteria and arachea against viruses. However, it has become a new approach for genome editing technology, being cheaper, quicker, and more accurate it has replaced the traditional methods such as meganuclease, zinc finger nuclease and TALEN (Transcripition Activator like effector nuclease). CRISPR-mediated genome targeting is performed by two components: single guide RNA and an endonuclease, Cas9 protein. CRISPR forms a complex with Cas9 and guides it to a specific DNA sequence creating a double stranded break (DSB). Various cellular DNA repair mechanisms cause desired insertions or deletions at target sites. This results in the modifications at the target sites that may be a knockout of a mutated gene, insertion of a new gene, either activation or repression of a gene in an individual. Thus, CRISPR-Cas9 is a simple and precise method used as a genomic tool having several applications in different fields. Its applications includes genome screening, treatment of various genetic disorders either by knockout or insertion of a gene, in agriculture, live imaging of genome, generation of animal models etc. Using a small RNA molecule of desired nucleotides (around-20bp) we can target specific genomes and edit the DNA of an individual with high specificity. Thus, CRISPR-Cas system has become a powerful tool in molecular biology as well as other fields within a small period of time. It has simplified the genome editing procedure with much more accuracy. Various experiments verifying the efficiency of CRISPR-Cas9 complex in precise editing of cells have been discussed in this report. It includes in-vitro cleavage of DNA and transfection of cancer cell line 084. Various techniques such as polymerase chain reaction and gel electrophoresis have also been discussed in this report.
Keywords: CRISPR, Cas9 protein, DSBs, NHEJ, HDR, PAM, sgRNA