CRISPR/Cas9을 이용한 어류 세포와 세균 유전자 타겟팅 및 그 발현 조절

Abstract

The bacterial adaptive immune system, CRISPR (clustered regularly interspaced short palindromic repeats), have spotlighted as a powerful and multi-purpose technology for genome editing. Recently, the CRISPR system expanded to the regulation of gene expression through the use of catalytically inactive dead Cas9 (dCas9); up-regulation through CRISPR activation (CRISPRa) and down-regulation through CRISPR interference (CRISPRi). The CRISPRi can interfere the expression of specific genes like customized DNA-binding proteins such as zinc-finger proteins or transcription activator-like effectors (TALEs) have been used as tools for sequence-specific DNA targeting and gene regulation. The nuclease-deficient Cas9, termed dCas9, guided by a single guide RNA (sgRNA) binds a target in genomic DNAwithout cleaving it, and the dCas9–sgRNA complex interfere with transcription elongation by blocking RNA polymerase (Pol) or transcription initiation by disrupting transcription factor binding. The CRISPRa system are expressed by using the dCas9 fused proteins can recruit transcription activators such as transcriptional activation domains (ADs). In natural systems, transcriptional initiation occurs through the coordinated recruitment of the necessary machinery by a number of locally concentrated transcription-factor ADs, so these ADs fusion protein can induce robust expression of target gene by the sgRNA targeting promoters region of the gene near transcription start sites (TSSs). In this study, By using the present CRISPR system and the application technologies, we want to check the regulation of target genes in fish cells, and differences of responses according to changed expression of targeted immune-related genes. The differences are also checked with cells knocked-out the genes by CRISPR-Cas9 system. We choose α subunit of Hypoxia-inducible factor-1 (HIF-1), which is key role in cellular homeostasis to hypoxia, as a target of CRISPRi system, and induced the down-regulation of the target gene in Epithelioma papulosum cyprini (EPC) cells. Additionally, in previous study the HIF-1α knock-out EPC cells showed the resistance to apoptosis and cytopathic effect (CPE) induced by Viral hemorrhagic septicemia virus (VHSV) more then normal EPC cells, so changes of susceptibility to VHSV in the EPC cell repressed expression of HIF-1α by CRISPRi system were analyzed with the EPC cells knocked-out HIF-1α gene. By the CRISPRa system, the activation of eGFP expression by targeting a CMV promoter in exogeneous plasmid vector was analyzed in EPC cells, and we induced the activation of Mx gene, known as antiviral protein, as endogenous target by targeting a promoter of the Mx gene in Hirame natural embryo (HINAE) cells. The repression of HIF-1α in EPC cells was analyzed through Quantitative real-time PCR (qPCR), and a robust and specific knockdown of HIF-1α was detected. The changes of susceptibility of VHSV in the EPC cells repressed expression of HIF-1α were analyzed through comparing the time of CPE observation with the HIF-1α knock-out EPC cells. The HIF-1α repression EPC cells showed the similar resistance to that of HIF-1α knock-out EPC cells. The activation of target genes was also analyzed through qPCR, and the expression of eGFP multiplied almost sixfold then control EPC, and the expression of Mx gene also multiplied more then 20-fold then control HINAE cells. By this study, the regulation of the target genes by the CRISPR application systems was confirmed in fish cells, and in further study these systems can utilize in several different ways by changing appropriate target of the systems to induce dramatic effect. We checked that the cells repressed the target gene by CRISPRi system showed similar response to the cells knocked out the gene, this result show a possibility to use the system for loss-of-function analysis.

The CRISPR-Cas9 system have developed as a genome editing technology, and productions of mutant in organism become relatively easier than that by past methods because of this technology. Because of this development, methods for the production of mutant bacteria was changed from complex traditional method such as using allelic exchange by conjugation to simple method; Using only a vector expressing CRISPR-Cas9 system and a donor template contained homologous arms for induction of Homologous directed recombination (HDR). In this study, we choose a gene of alanine racemase (alr), an enzyme catalytic for change L-alanine to D-alanine that is essential for cell wall synthesis in bacteria, in Escherichia coli (E. coli) as a target of CRISPR/Cas9 system, and induced the mutation of alr gene in E. coli by this system. The production of mutant strain knocked out alr gene was performed by transformation of vectors expressing CRISPR/Cas9 system and containing donor template targeting alr gene, and the changes of alr gene sequence in E. coli was detected by PCR and sequencing of the gene. Conclusionally, the mutation of E. coli was checked by sequencing of alr gene to equal donor template design, and we checked the successful production of induced mutant strain of E. coli by the system. This system can be extended out pathogenic bacteria in aquaculture, and enable to induce mutation easily such as the auxotrophic mutant, and at a further study, the mutant would be used as a attenuated vaccine.