Chlorella species are indispensable microalgae for biorefinery but are hardly in DNA manipulation due to the high guanine-cytosine (GC) contents of DNA. In this study, we established a new approach via 20 guanines for sgRNA design, which is annotated as “Adaptive Single Guide Assisted Regulation DNA (ASGARD)” and coupling with CRISPR interference associated dCas9 system to overcome the difficulties. At first, C. sorokiniana was predominate as its faster growth rate when compared to C. vulgaris and C. variabilis in the culture using Tris-acetate-phosphate (TAP) medium. Among all the genetic transformants, gene regulation via CRISPRa-VP64 (CRISPRa) enhanced the protein contents up to 60% (w/w) of dry cell weight, where the highest concentration was 570 mg/L. Meanwhile, CRISPRi-KRAB (CRISPRi) with ASGARD increased protein content to 65% and lipid formed in the range of 150 mg/L to 250 mg/L. From the transcriptome analysis, we deciphered 468 genes down-regulated and 313 genes up-regulated via CRISPRi, while less difference existed in CRISPRa. This novel design and technology reveal a high potential of gene-regulating approach to other species for the biorefinery and bio-industry.

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