PROKARYOTIC ENZYME SYSTEM CRISPR/CAS9 IN GENOME EDITING OF CIONA INTESTINALIS
ILIANA A. IVANOVA1*, MANOELA ANDREEVA, YULIA KUTZOVA, ЕLENA NENOVA2
1-Department of General and industrial microbiology, Faculty of Biology, 8 Dragan Tzankov, Sofia University St. Kliment Ohridski, Sofia, Bulgaria
2-Department of Zoology and anthropology, Faculty of Biology, 8 Dragan Tzankov, Sofia University St. Kliment Ohridski, Sofia, Bulgaria
*Corresponding author: ilivanova@abv.bg
Keywords: target genes editing, horde animals, embryos
Abstract: Excluding genes with CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 is a new approach to studying the functions of genes in different organisms.
Cas9 enzyme is isolated from the prokaryotic organisms and is used as molecular scissors for cutting of specific regions of DNA. Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms.
The CRISPR method consist of a highly effective set of molecular "scissors" - can be easy to use but is not perfect. These "scissors" can cut more than necessary by cutting DNA into unexpected and unwanted sites. In early experiments, scientists have found that side effects can occur at some sites in DNA at approximately the same frequency as the target areas. This method includes three major components: the Cas9 enzyme that cuts a portion of the DNA; single-stranded RNA sequence (a series of nucleotides in its molecule) that gives Cas9 the exact place to cut; a new template DNA to repair the cut ("repair" is a DNA double strand recovery). Using CRISPR/Cas9, double-strand fractures are induced in the genome of Ciona intestinalis. CRISPR/Cas9 can mutate endogenous Ciona intestinalis genes, a great model for clarifying molecular mechanisms for building a horizontal plan of the body. CRISPR/Cas9 is sufficiently effective and specific to generate a large number of embryos carrying mutations in the target gene of interest, which allows rapid screening of the gene in Ciona intestinalis.