Data_Sheet_1_Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method.docx

Chlamydomonas reinhardtii is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic value of C. reinhardtii. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in C. reinhardtii. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9-mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene CrFTSY was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knock-out phenotype of CrFTSY and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in C. reinhardtii. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of C. reinhardtii.</p

Putative biomineralization-related genes in DEGs at different calcium concentrations.

The log2 fold changes of the genes are shown as a bar chart (log2FC; ± SE, light gray: [Ca2+] 0.1 mM relative to [Ca2+] 0 mM, dark gray: [Ca2+] 10 mM relative to [Ca2+] 0.1 mM, black: [Ca2+] 10 mM relative to [Ca2+] 0 mM). The gene IDs in the bar chart are in the following order: SLC4-2 (c31857_g11_i1), SLC family (c25647_g1_i1), Na+/H+ exchanger (c31114_g3_i2), delta CA (c24914_g1_i2), calcium-binding GPA (glutamic acid, proline and alanine) protein (c20552_g1_i1), Ca2+/Mg2+ permeable cation channels (LTRPC family) (c30953_g1_i11), fibrillins and related proteins containing a Ca2+-binding epidermal growth factor (EGF)-like domain (JGI ID 118025; c28155_g1_i1), fibrillins and related proteins containing Ca2+-binding EGF-like domain (JGI ID 463266; c28980_g1_i1), hypothetical protein (c34539_g1_i1), eukaryotic initiation factor 4A (eIF4A) (c22513_g1_i1), ABC transporter (c34539_g1_i1), and putative mitochondrial chaperone BCS1 (c23862_g2_i1). Asterisks represent significant expression change (*p < 0.05, **p < 0.005).</p

KEGG pathway enrichment of DEGs at different calcium concentrations.

The pathways enriched in (A) up-regulated and (B) down-regulated DEGs in the comparison between [Ca2+] 10 vs 0 mM and [Ca2+] 10 vs 0.1 mM.</p

Unigenes differentially expressed in both the transcriptome and proteome.

(A) The differentially expressed genes (DEGs) and proteins (DEPs) in [Ca2+] 10 mM relative to [Ca2+] 0.1 mM (yellow: up-regulated, blue: down-regulated). (B) The putative biomineralization-related DEPs (log2FC; ± SE): AEL1 (c31742_g1_i2), calcium pump (c31573_g1_i1), mitochondrial chaperone BCS1 (c23862_g2_i1), and eIF4A (c22513_g1_i1). (C) Correlation between differentially expressed transcripts and proteins.</p

Summary of RNA-sequencing and <i>de novo</i> assembly.

Summary of RNA-sequencing and de novo assembly.</p

KOG enrichment analysis of DEGs compared with the transcriptome.

The up-regulated DEGs in [Ca2+] 10 mM relative to [Ca2+] 0 mM were analyzed.</p

Data_Sheet_2_Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method.docx

Chlamydomonas reinhardtii is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic value of C. reinhardtii. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in C. reinhardtii. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9-mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene CrFTSY was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knock-out phenotype of CrFTSY and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in C. reinhardtii. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of C. reinhardtii.</p

Annotation of unigenes against public databases.

Annotation of unigenes against public databases.</p

The growth rate and Fv/Fm measurements at different calcium concentrations.

The growth rate and Fv/Fm measurements at different calcium concentrations.</p

KEGG pathways in DEGs and DEPs of <i>E</i>. <i>huxleyi</i> at different calcium concentrations.

(A) The up- and (B) down-regulated DEGs and DEPs involved in metabolic pathways of E. huxleyi. The numbers of common and unique DEGs and DEPs are shown in the Venn diagram. The bar chart shows the sum of the pathways commonly involved in DEGs and DEPs.</p