MOESM1 of Heterologous and endogenous U6 snRNA promoters enable CRISPR/Cas9 mediated genome editing in Aspergillus niger
Creators: name: Zheng, Xiaomei
name: Zheng, Ping
name: Sun, Jibin
name: Kun, Zhang
name: Ma, Yanhe
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Inorganic Chemistry
subject: FOS: Chemical sciences
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology

description: Additional file 1. Figure S1. qPCR results of sgRNA expression levels from each promoter. Total RNA was isolated, converted to cDNA, and sgRNA expression level was quantified. sgRNA expression levels were normalized to the amount of sgRNA generated by PhU6 promoter. 18S rRNA was used as internal control. Bars represent the fold change of sgRNA level under the control of different U6 promoters (mean ± SD; n = 3). Figure S2. Transformants with albA disruption by inserted the donor DNA with short homologous arms. Transformants XM6 grew on the primary transformation plates after co-transformed pCas9, sgRNA3.1 and donor DNA MHi-albA-hph. Transformants NC1 grew on the primary transformation plates after only co-transformed pCas9 and donor DNA MHi-albA-hph. Transformants NC2 grew on the primary transformation plates after only co-transformed sgRNA3.1 and donor DNA MHi-albA-hph. Figure S3. DNA sequencing analyses for genetic context at the DSBs in albA gene inserted transformants XM6. DNA sequencing results of PCR products amplified by albA-g-F/hph-R (a) and hph-F/albA-g-R (b) using the genomic DNA of albino colonies XM6 as templates. The red letters represent the protospacer sgRNA-albA1, and the yellow shaded red letters represent the PAM site. The green letters represent the to-be-inserted hph cassette, and blue letters represent the homology arms in the donor DNA MHi-albA-hph. XM6.1-6.11 represent the selected albino colonies. Table S1. A. niger strains used in this study. Table S2. Plasmids used in this study. Table S3. Primers used in this study. Restriction sites are underlined. Fm represents forward primer with modification and Rm represents reverse primer with modification. The modified additional sequences were represented in lowercase letters. Table S4. DNA sequences of codon optimized cas9 used in this study. Black letters indicate the codon-optimized cas9 gene. Purple letter indicate the NLS sequences of SV40 at 5’-termini and nucleoplasmin at 3’-termini. Green letters indicate the gfp (S65T) gene from pMF272. Orange letters indicate the (G4S)3 linker sequence. Table S5. DNA sequences of sgRNA constructs used in this study. Green letters indicate the promoter region for sgRNA expression. Orange letters indicate the transcription start of U6 promoters. Blue letters indicate the sgRNA scaffold. Blue underlined letters indicate BbsI restriction sites. Red letters indicate the terminator of RNU6 gene. Red underlined letters indicate genetic targets. Table S6. DNA sequences of donor DNA used in this study. Blue letters indicate the homogenous arms located at the 5’ and 3’ flanking region of the genetic target sites. Black lowercase letters indicate the selection marker cassettes.