Novel Microbial Modification Tools to Convert Lipids into Other Value-Added Products
CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) is a microbial adaptive immune system that has revolutionized the field of molecular biology and genome engineering. The Type II CRISPR system consists of Cas9 nuclease of Streptococcus pyogenes and the RNA complex that guides Cas9 nuclease to a specific sequence of DNA in the genome. The CRISPR-Cas9 technology has reformed our ability to edit DNA and to regulate expression levels of genes of interest to high precision and accuracy. It is a powerful technology, which is used for genome engineering of a wide range of organisms for various applications. Here, we describe a method involving CRISPR-Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms for biotechnological applications in yeast. The complete procedure of genome editing including target sequence selection, cloning gRNA with a target sequence, transformation, and verification of the desired mutation/deletion or insertion can be achieved within 2–3 weeks in yeast.
Key wordsCRISPR-Cas9 gRNA CRISPR RNA Trans-activating CRISPR RNA Genome editing Homology-directed repair Nonhomologous end joining
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