CRISPRa/i with Adaptive Single Guide Assisted Regulation DNA (ASGARD) mediated control of Chlorella sorokiniana to enhance lipid and protein production

Abstract

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‐1. Meanwhile, CRISPRi‐KRAB (CRISPRi) with ASGARD increased protein content to 65% and lipid formed in the range of 150–250 mg L‐1. 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.

Recent advances and fundamentals of microalgae cultivation technology

“…The U.S. Food and Drug Administration (FDA) authorized C. reinhardtii microalgae for human consumption, and it was widely utilized for the manufacture of protein. [176][177][178] Genetic modification of this strain allowed it to effectively accumulate zeolin (chimeric recombinant protein of zein and phaseolin, the major seed storage proteins from maize and beans), thus creating a promising platform for protein production. [66] Remarkably, compared to individual proteins, the zein and phaseolin chimera have a better balanced amino acid composition.…”

Harnessing genetic engineering to drive economic bioproduct production in algae

“…The Cas9/single guide RNA (sgRNA) RNP delivery enhanced the HDR at the nitrate reductase (NR) target site, generating ∼70% of positive mutant lines, indicating a significant improvement in editing efficiency compared to the native HDR system alone. Additionally, Lin et al (2021) developed a novel approach to designing sgRNA via 20 guanines, called Adaptive Single Guide Assisted Regulation DNA (ASGARD) and coupled it with the dCas9 system in C. sorokiniana . Among the transformants, this approach led to an increase in protein content, reaching up to 60% (w/w) of DCW, with the highest protein concentration being 570 mg/L ( Lin et al, 2021 ).…”

“…Additionally, Lin et al (2021) developed a novel approach to designing sgRNA via 20 guanines, called Adaptive Single Guide Assisted Regulation DNA (ASGARD) and coupled it with the dCas9 system in C. sorokiniana . Among the transformants, this approach led to an increase in protein content, reaching up to 60% (w/w) of DCW, with the highest protein concentration being 570 mg/L ( Lin et al, 2021 ). In another study, the authors developed a CRISPR-Cas9 reverse-genetics pipeline and used it to identify a TF that regulates lipid accumulation, called ZnCys, in Nannochloropsis gaditana (CCMP 1894) ( Ajjawi et al, 2017 ).…”

CRISPR-based bioengineering in microalgae for production of industrially important biomolecules

“… Lin et al (2022) used CRISPRa/i system to modulate the gene expression in Chlorella sorokiniana UTEX 1602. They demonstrated that gene regulation via dCas9-VP64 (CRISPRa) increased the protein contents by approximately 60% (w/w), while that via dCas9-KRAB (CRISPRi) increased the protein content to 65%, and lipid accumulated in the range of 150–250 mg/L (WT: 150 mg/L).…”

“…The mutants were negatively selected on potassium chlorate or 2-fluoroadenine clearly exhibiting the practicality of generating markerless mutants in Chlorella that might likely not fall under regulatory purview (Kim et al, 2021). Lin et al (2022) used CRISPRa/i system to modulate the gene expression in Chlorella sorokiniana UTEX 1602. They demonstrated that gene regulation via dCas9-VP64 (CRISPRa) increased the protein contents by approximately 60% (w/w), while that via dCas9-KRAB (CRISPRi) increased the protein content to 65%, and lipid accumulated in the range of 150-250 mg/L (WT: 150 mg/L).…”