Establishment of CRISPR system ribonucleoprotein complexes (Cas9-RNP) for seamless genome editing in Chlamydomonas reinhardtii

Abstract

Cas9-associated ribonucleoproteins (Cas9-RNPs) is a novel application of CRISPR system and can achieve seamless DNA editing which different from previous strategy that delivering nuclease of gene-editing function into host cells instead of expressed by itself then functioning Three part are mainly contained in this study First establishment of a dual plasmid system which co-express Cas9 protein and single guide RNA (sgRNA) Second analysis of gene cleavage functions by in vitro test Finally mediating Cas9-RNP into microalgae Chlamydomonas reinhardtii (C reinhardtii) CC400 for seamless gene editing and improving production of cell metabolites by in vivo test First of all a common-used plasmid pSG-RSF with high copy number was constructed Afterwards sgRNA which target on Ribulose-1 5-bisphosphate carboxylase (rbcL) gene was inserted into the pSG-RSF It was ascertained that His-tag protein fusion at N-terminus is more helpful in increasing recovery rate of purification than fusion at C-terminus as well to decrease production cost Average of 200 ?g Cas9-RNP was produced from each 50 mL E coli cultured medium after purification by nickel ion affinity chromatography Hereafter Cas9-RNP capability of gene cleavage was validated by in vitro test with utilization of amplified fragment that PCR from C reinhardtii CC400 genome Analysis result represented that activity performed stable when reaction times at 1 2 4 8 hours and within produced 21 days Additionally a gene element ‘RiboJ’ which can enhance expression level is first investigated in this study for improving Cas9 protein expression in CRISPR system However negative result was observed for enhancing protein expression due to length up to 4107 bp of Cas9 gene fragment as other gene fragments from about 600 bp to 2700 bp were enhanced in contrast Results illustrated that open reading frame (ORF) of regulated genes are limited below certain length and RiboJ is not suitable for enhancing Cas9 express cassette driven by a weak promoter Finally we introduced Cas9-RNP that mediate phytoene synthase (psy) gene in C reinhardtii CC400 to regulate metabolic flux in carotenoid pathway under the optimized electroporation conditions as a 180 V poring pulse followed by four 20 V transfer pulse with 20 ?g of Cas9-RNP added After cultivated in liquid medium with 100 mg/L ampicillin phenotype of CC400 strain through gene editing represented light-green color which represented no distinction on cell growth compare to wild type (WT) strain It can be speculated that Cas9-RNP does affect the metabolic pathway of algal pigment production Different form of detection peak from WT has been discovered after analysis by high-performance liquid chromatography (HPLC) Therefore it was validated that alteration of carotenoids metabolic flux forced by psy gene editing with Cas9-RNP In conclusion it elucidates that programmable Cas9-RNP is suitable for seamless gene editing in CC400 Moreover the off-target effect can be decreased as well as the toxicity problem derived from long period expression of Cas9 protein can be solved due to Cas9-RNP degrade by itself which won't allow it to long-term stay in host cell

Date of Award	2019
Original language	English
Supervisor	I-Son Ng (Supervisor)