Recombinant DNA-based molecular techniques CRISPR-CAS9 2020-2021 PDF

Recombinant DNA-based molecular techniques (part III) CRISPR-CAS9 and gene editing Prof. Mamoun Ahram What is CRISPR/Cas9? CRISPR is clustered regularly interspaced short palindromic repeats It is a bacterial genetic system that constitutes the immune system of bacteria against phages. Cas9 is a RNA...

Recombinant DNA-based molecular techniques (part III) CRISPR-CAS9 and gene editing Prof. Mamoun Ahram What is CRISPR/Cas9? CRISPR is clustered regularly interspaced short palindromic repeats It is a bacterial genetic system that constitutes the immune system of bacteria against phages. Cas9 is a RNA-guided nuclease that can either create single or double strand breaks. The nuclease is directed to its target sequence by a short RNA fragment known as a guide RNA (gRNA) or single guide RNA (sgRNA), which is complementary to the target segment of the genome. The concept When a phage infects a bacterial cell, the cell degrades the phage DNA into smaller pieces and integrates one of these fragments into the CRISPR cluster. When the phage infects the cell again, the cell transcribes the DNA into RNA (guide RNA or gRNA), which is integrated into the Cas9 nuclease and guides it to the phage DNA to degrade it. In 2020… Emmanuelle Charpentier and Jennifer Doudna DNA repair mechanisms in human cells The consequences of DNA damage repair The steps of action Both the gRNA and Cas9 gene can be introduced into human cells as genes cloned into plasmid vectors. Gene editing Normal Through either mechanism, the function of a gene can be studied by mutating it. This DNA is introduced into cells so the DNA repair mechanism uses it for recombination. Specifically in this mechanism, a mutated gene is replaced by a normal one (or the opposite). Other creative uses Transcriptional regulatory factors can be added to a “dead” Cas9 (dCas9), enabling them to turn genes on or off or adjust its level of activity. GFP can be added to visualize genes. Other Cas enzymes Cas12a: A smaller enzyme that introduces staggered cut. Cas13a: A RNA endonuclease Controversial issue Gene repair https://www.theguardian.com/science/2016/ jan/13/uk-scientists-ready-to-geneticallymodify-human-embryos The dark side of science https://www.theguardian.com/wo rld/2019/dec/30/gene-editingchinese-scientist-he-jiankui-jailedthree-years Bioterrorism Bioterrorism