Cas9-mediated Stearoyl-CoA desaturase 1 (SCD1

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Biotechnology and Biological Transformations

CRISPR/Cas9-mediated Stearoyl-CoA desaturase 1 (SCD1) deficiency affects fatty acid metabolism in goat mammary epithelial cells Huibin Tian, Jun Luo, Zhifei Zhang, Jiao Wu, Tianying Zhang, Sebastiano Busato, Lian Huang, Ning Song, and Massimo Bionaz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03545 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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Journal of Agricultural and Food Chemistry

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CRISPR/Cas9-mediated Stearoyl-CoA desaturase 1 (SCD1) deficiency affects

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fatty acid metabolism in goat mammary epithelial cells

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Huibin Tian†, Jun Luo†*, Zhifei Zhang†, Jiao Wu†, Tianying Zhang†, Sebastiano

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Busato#, Lian Huang†, Ning Song†, Massimo Bionaz#*

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†Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal

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Science and Technology, Northwest A&F University, Yangling, 712100, China

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#Department of Animal and Rangeland Sciences, Oregon State University, Corvallis

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97331, USA

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*Corresponding authors: [email protected]. and

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[email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing the cis

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double bond formation in ∆9 position to produce monounsaturated fatty acids

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essential for the synthesis of milk fat. Previous studies using RNAi methods have

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provided support for a role of SCD1 in goat mammary epithelial cells (GMEC);

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however, RNAi present several limitations that might preclude a truthful

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understanding of the biological function of SCD1. To explore the function of SCD1

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on fatty acid metabolism in GMEC, we used CRISPR/Cas9-mediated SCD1 knockout

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through NHEJ (Non-Homologous End-Joining) and HDR (Homology-Directed

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Repair) pathways in GMEC. We successfully introduced nucleotides deletion and

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mutation in the SCD1 gene locus through NHEJ pathway, and disrupted its second

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exon via insertion of an EGFP-PuroR segment using HDR pathway. In clones derived

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from the latter, gene and protein expression data indicated that we obtained a

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monoallelic SCD1 knockout. A T7EN1-mediated assay revealed absence of no

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off-targets in the surveyed sites. The content of triacylglycerol, cholesterol and

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desaturase index were significantly decreased as consequence of SCD1 knockout. The

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deletion of SCD1 decreased the expression of other genes involved in de novo fatty

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acid synthesis, including SREBF1 and FASN, as well the fatty acid transporters FABP3

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and FABP4. The downregulation of these genes partly explains the decrease of

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intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat

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mammary cells using CRISPR/Cas9. The demonstration of the successful use of

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CRISPR/Cas9 in GMEC is an important step to produce transgenic goats to study 2


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mammary biology in vivo.

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KEYWORDS: CRISPR/Cas9, SCD1, NHEJ, HDR, goat mammary epithelial cells

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INTRODUCTION

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Dairy goats are of increasing global importance among grazing livestock, with

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steadily growing populations, especially among developing countries1, 2. Furthermore,

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goat milk has been under the spotlight in recent years3, 4, for it is a significant source

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of short-chain fatty acids and monounsaturated fatty acids (MUFA) with reported

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beneficial roles in human health5. The formation of the MUFA palmitoleate (16:1 n-7)

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and oleate (18:1 n-9) relies on the activity of stearoyl-CoA desaturase 1 (SCD1), a

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∆9-fatty acyl CoA desaturase that catalyzes the synthesis of double bonds in

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cis-delta-9 position of long-chain fatty acids6. In lactating ruminants, the expression

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of mammary SCD1 increases dramatically concomitantly with a large increase in fatty

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acid synthesis during lactation7, 8. The important role of SCD1 in triacylglycerols

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synthesis and fatty acid composition was recently confirmed in goat mammary

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epithelial cells (GMEC) using a combination of gene overexpression and gene

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silencing techniques9. The same study also identified SREBP1 and PPARγ as

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important transcriptional regulators of SCD1. A partial loss-of-function approach,

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such as the use of RNA interference (RNAi) can offer insight on gene function;

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however, this technique presents several limitations, including low and inconsistent

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efficiency of gene silencing10, and short-term inhibition of gene expression11.

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Furthermore, RNAi does not allow to conduct studies when specific gene and protein

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dosages are crucial12. Studies of the transcriptional and biological role of SCD1 would

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undoubtedly benefit from more consistent, long term knockout models.

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A permanent loss-of-function model might be achieved through the novel 4


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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated

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gene editing technique. CRISPR has been used successfully in a variety of species,

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such as bacteria, yeasts, fruit flies, mice and monkeys13-16. Because of its low cost,

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great flexibility, and high efficiency in genome editing, it has been applied for

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generating gene modified animal models of human diseases17, 18, to obtain transgenic

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pigs19 and to perform in vivo study of long non-coding RNA20. CRISPR models

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utilize an endogenous endonuclease (such as the CRISPR-associated protein 9, or

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Cas9) to cause a DNA double strand break (DSB) at a specific site, to which the

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nuclease is directed by a single-guide RNA (sgRNA). The cells have the ability to

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repair their DNA21 by two main pathways: the error-prone Non-Homologous

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End-Joining (NHEJ), which can cause random nucleotide insertion, deletion and

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mutation22, inducing a frame shift and a subsequent early stop codon; or the more

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accurate Homology-Directed Repair (HDR)23, which integrates a homologous DNA

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sequence (i.e. a sister chromatin or a plasmid)24, 25.

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While global26, 27 or tissue specific knockout28-30 have been carried out to study

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the physiological function of SCD1 in mice, no evidence of such endeavors exists in

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goats. Compared to mice, the identification of the role of SCD1 in lactating dairy

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goats is of greater importance: MUFA in goat milk are mostly obtained via

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endogenous desaturation, considering that fatty acid biohydrogenation in ruminant

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animals markedly decreases diet-sourced unsaturated FA31.

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This study aims at providing crucial insight on the transcriptional regulation of

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SCD1 and its role in milk fatty acid synthesis using a CRISPR/Cas9-mediated 5



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knockout model, through both the NHEJ and HDR repair pathways in GMEC.

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MATERIALS AND METHODS

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Ethics Statement. All the experimental procedures were carried out in accordance

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with the Institutional Animal Care and Use Committee in the College of Animal

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Science and Technology, Northwest A&F University, Yang Ling, China (permit

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number: 15-516, date: 2015-9-13).

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Cell culture and suitable concentration of puromycin. GMEC were isolated from

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five Xinong Saanen dairy goats at peak lactation (60 d after parturition) as described

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previously32, and were purified and cultured according to our previously described

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protocol33. Briefly, GMEC were isolated by tissue block preparation technique. The

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mammary gland tissue was cut into about 1mm3. The tissue block was cultured in 5%

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CO2 at 37℃ with basal medium. The culture medium was changed every two or three

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days until the epithelial cells separated from the tissue block. Then the cells were

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digested from tissue block. The basal culture medium contained DMEM/F12 medium

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(11320-033, Invitrogen Corp., Waltham, MA, USA), 10% fetal bovine serum

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(10099-141, Invitrogen), 5 µg/mL bovine insulin (16634, Sigma, St. Louis, MO), 5

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mg/L hydrocortisone (H0888, Sigma), 100 U/mL penicillin/streptomycin (080092569,

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Harbin Pharmaceutical Group, Harbin, P. R. China), and 10 ng/mL epidermal growth

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factor (PHG0311, Invitrogen). GMEC were incubated in 5% CO2 at 37℃ and

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medium was changed every 24 hours. To promote lactogenesis, the cells were

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cultured in the basal medium with prolactin (L6520, 2 µg/mL, Sigma) for 48 h before

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performing the following experiments. 6


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GMEC were seeded in six-well plate, and when the cells approached to 80-90%

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confluence, puromycin (P8833, Sigma) was added to the culture medium at 0, 0.5, 1.0,

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or 1.5 µg/mL concentration. After cultured for 72 hours, the cell death condition was

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examined by microscope. The lowest lethal dose of puromycin to the GMEC was 1.0

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µg/mL.

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Construction of Cas9/sgRNA expression vector and HDR donor vector. The

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sgRNAs which targeted to exon 2 of Capra hircus SCD1 were designed using the

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online

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(http://chopchop.cbu.uib.no/)34. Three sgRNAs (Figure 1A) were selected based on

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their predicted score and lower off-target effects for Cas9/sgRNA expression vector

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construction. For the NHEJ-mediated pathway, we chose the all-in-one vector

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pSpCas9 (BB)-2A-Puro (plasmid#62988, PX459 V2.0, Addgene, MA, USA), a gift

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from Feng Zhang35. The sgRNAs were synthesized as single-strand DNA

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oligonucleotides by Invitrogen (Shanghai, China), and annealed oligonucleotides were

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inserted into PX459 vector containing two BbsⅠ (R3539S, NEB, Ipswich, MA, USA)

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enzyme sites according to a previous protocol35. The sgRNA with higher efficiency

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was used for the single clone selection in both NHEJ- and HDR-mediated genome

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editing.

CRISPR

design

tools

(http://crispr.mit.edu/)

and

CHOPCHOP

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For HDR-mediated genome editing, pX330-U6-Chimeric_BB-CBh-hSpCas9

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(plasmid#42230, PX330, Addgene, MA, USA, a gift from Feng Zhang) was used as

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the Cas9/sgRNA co-expression plasmid36, inserting a sgRNA using the two BbsⅠ

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enzyme sites. The donor vector (a gift from Key Laboratory of Animal Biotechnology, 7



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Ministry of Agriculture, Northwest A&F University), used to deliver the homology

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arm, contained the coding sequence of puromycin resistance and enhanced green

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fluorescent protein (eGFP) as selection markers. These two genes were fused through

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the porcine teschovirus-1 2A (P2A) peptide sequence37, which allows co-expression

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of the two constructs.

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Genomic DNA was extracted from blood samples of five goats using a Universal

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Genomic DNA kit (CW2298S; CW Biotech, Beijing, China). A 1193 bp 5’

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homologous arm fragment and 1063 bp 3’ homologous arm fragment were amplified

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by PCR using PrimeSTAR® Max DNA Polymerase (R045A, Takara Bio Inc., Otsu,

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Japan) according to the manufacturer’s protocol. The reaction was performed at 98℃

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for 10 s, followed by 55℃ for 5 s, and 72℃ for 10 s. 5’ arm and 3’ arm clone primers

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are shown in Table 1. The PCR products were sequenced by Invitrogen (Shanghai,

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China). Then 5’ arm and 3’ arm fragments were inserted into the donor vector. Two

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LoxPs in the same orientation were inserted around the selection markers, just in case

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they were required to be removed38. A schematic representation of the donor vector

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can be observed in Figure 2A.

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Plasmid transfection, DNA extraction and T7EN1 assay. GMEC were cultured in

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six-well plates to 70-80% confluence. For the NHEJ pathway, 2µg PX459 was

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transfected using Lipofectamine™ 2000 (11668019, Invitrogen, Waltham, MA, USA)

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according to the manufacturer’s protocol. Forty-eight hours after transfection, the

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cells were cultured in basal medium with puromycin for four to five days. Viable cells

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were collected for genomic DNA extraction using a Universal Genomic DNA Kit 8


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(CWBIO, China). The genomic region flanking the target site was PCR amplified

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using the test primers for NHEJ (Table 1). The PCR products were purified by PCR

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Clean-Up Kit (AP-PCR-50, Axygen, CA, USA) according to the manufacturers’

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instructions. Purified DNA was annealed for T7EN1 cleavage assay39 (M0302L, NEB,

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Ipswich, MA, USA) and the enzyme digestion product was analyzed by agarose gel

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electrophoresis. Cleaved bands intensity were measured by ImageJ software

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(ImageLab, http://imagej.net). The frequency of PCR product enzyme digestion fcut

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was determined by the formula (a+b)/(a+b+c), where a is the intensity of the

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undigested PCR product and b and c are the intensities of each cleavage bands. The

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indel occurrence in the DNA was estimated based on the binomial probability

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distribution of duplex formation as following: Indel % = (1 − (1 − )) ∗ 100

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Then PCR products were cloned into a pMD19-T vector and sequenced by Invitrogen (Shanghai, China) to assess sequence modification in the cell pools.

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For the HDR pathway, 1 µg of donor vector together with 1 µg of PX330 were

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transfected into GMEC using Lipofectamine™ 2000 (Invitrogen, USA). Puromycin

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selection was performed 48 h after transfection for four to five days until co-incubated

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wild-type cells, treated with the same antibiotic concentration, had 100% mortality. A

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separate batch of cells, transfected with the donor vector and a PX330 that did not

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contain any sgRNA, were used as a control.

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Detection of individual GMEC clones by PCR and off-target analysis. After

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selection, cells were counted using a hemocytometer and diluted to a final 9



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concentration of 1 cell/100 µL. Individual cells were then plated in 96-well plates and

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cultured for 10-14 days to obtain single clone colonies. The medium for the cell

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culture was the basal culture medium containing DMEM/F12 medium, 10% fetal

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bovine serum, 5 µg/mL bovine insulin, 5 mg/L hydrocortisone, 100 U/mL

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penicillin/streptomycin, and 10 ng/mL epidermal growth factor. Cells from each

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colony were collected by trypsinization. Half of the cells were plated in the 48-well

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plates, and the remaining cells were collected in a 1.5 mL tube, and 1 mL PBS buffer

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was used to resuspend the cells and then the cells were centrifuged for 5 minutes at

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400×g to discard the PBS buffer. This process was performed for three times to wash

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the cells and cells were resuspended in lysis buffer (10 mM Tris-HCl; 50 mM KCl;

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1.5 mM MgCl2; 0.5% Tween-20; 100 ng/µL proteinase K) for PCR analysis. The

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lysate was incubated at 56℃ for 45 min and then at 95℃ for 10 min. For PCR

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analysis, 2 µL of the cell lysate was added to the PCR reaction. For the

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NHEJ-mediated knockout, test primers for NHEJ (Table 1) were used to amplify the

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region including sgRNA target sites, while for the HDR-mediated knockout, 5’

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junction primer and 3’ junction PCR primer (Table 1) were employed, and the PCR

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strategy is shown in Figure 2A. The reaction conditions were as follows: 98℃ for 10 s,

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55℃ for 5 s, and 72℃ for 5 s using PrimeSTAR® Max DNA Polymerase (Takara).

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Mutations in the HDR single clone was also confirmed by measuring eGFP

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fluorescence using a Leica fluorescent microscope (DMI4000B, Wetzlar, Germany).

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Wild type cells were used as control under the same exposure and filter settings.

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Off-target (OT) sites were predicted using the online website tool Cas-OFFinder 10


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(http://www.rgenome.net/cas-offinder/)40. Mismatches ≤ 4 bp was used as criteria41, 42.

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Genomic DNA extracted from both HDR single clone and NHEJ single clone were

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used as templates for off-target sites PCR. T7EN1 assay was used and the PCR

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product were inserted into pMD19-T vector for sequencing. The primers for off-target

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detection are shown in Suppl. Table 2.

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RNA extraction and real-time quantitative PCR (RT-qPCR). Total RNA was

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extracted from GMEC with RNAiso Plus (9109, Takara) according to the

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manufacturer’s protocol and 0.5 µg of total RNA was used to synthesize cDNA with

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PrimeScript® RT Reagent Kit with gDNA Eraser (RR047A, Perfect Real Time,

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Takara). cDNA was diluted to 400 ng/uL for each sample. RT-qPCR were performed

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using SYBR Premix Ex Taq Ⅱ (RR820A, Perfect Real Time, Takara). All the primers

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used for RT-qPCR are listed in the Suppl. Table 1. The real-time PCR reactions were

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performed in a CFX-96 Real-Time PCR Detection system (Bio-Rad Laboratories Inc.,

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Hercules, CA). The reactions were performed as 95℃ for 30 s, followed by 40 cycles

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of 95℃ for 5 s and 60℃ for 30 s; a dissociation curve was performed at 95℃ for 10 s

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and then from 65℃ to 95℃ with a 0.5℃ increase. The RDML files were used to

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calculate the RT-qPCR values for each gene and sample using LinRegPCR43. Four

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transcripts were tested as potential internal controls or reference genes using

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geNorm44, these were ribosomal protein S9 (RPS9), ribosomal protein S15 (RPS15),

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mitochondrial ribosomal protein L39 (MRPL39), and ubiquitously expressed

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transcript (UXT)45. The normalization factor was calculated using the expression of

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RPS9, MRPL39, and UXT for both HDR and NHEJ experiment. The V-value using 11



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these three genes was < 0.10 for both experiments45.

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Protein extraction and western blot analysis. Cells were collected and lysed in

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ice-cold RIPA buffer (R0010, Solarbio, Beijing, China) with protease inhibitor

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(04693132001, Roche Diagnostics Ltd, Mannheim, Germany). Protein concentration

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was measured using BCA protein assay kit (23227, Thermo Fisher Scientific,

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Rockford, IL). The protein was separated with 10% SDS/PAGE, transferred onto

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nitrocellulose membrane (HATF00010, Millipore, Massachusetts, USA) by a Bio-Rad

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Trans-Blot SD semi-dry transfer cell (Bio-Rad Laboratories Inc., Hercules, CA, USA),

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and the membranes were blocked for 1.5 h using 5% skim milk (232100, BD,

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Franklin Lakes, New Jersey, USA). The membranes were incubated overnight at 4℃

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with primary antibodies for SCD1 (Cat#ab39969, Abcam, Cambridge, MA, 1:300)9,

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SREBP1 (Cat#ab3259, Abcam, Cambridge, MA, 1:1000)46and β-actin (CW0096,

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1:1000; CW Biotech, Beijing, China). After the membranes were washed with TBST

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for three times, horseradish peroxidase (HRP)-conjugated goat anti-rabbit (CW0103,

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1:2000; CW Biotech) and goat anti-mouse IgG (CW0102, 1:2000; CW Biotech) were

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used as secondary antibodies. Signals were measured using an enhanced

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chemiluminescent (ECL) Western blot system (1705061, Bio-rad). The density of the

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bands was analyzed by Image J and the relative expression of protein was normalized

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to β-actin.

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Measurement of total cellular triacylglycerol (TAG) and cholesterol. Cells were

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plated in 60 mm culture dishes and, upon reaching 90% confluence, cellular TAG and

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cholesterol were detected with GPO-Trinder triacylglycerol assay kit (E1013, 12


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Applypen Technologies Inc., Beijing, China) and cholesterol assay kit (E1015,

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Applygen Technologies Inc.) as described previously47. In brief, culture medium was

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discarded, and cells were washed for three times using PBS buffer. Then GMEC were

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treated with lysis buffer for 10 minutes and cells were scraped. The supernatant was

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collected by centrifugation for TAG and cholesterol assay at 550 nm using a Biotek

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microplate reader (Winooski, VT, USA). The amount of total protein was detected

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using BCA protein assay kit (Thermo Fisher Scientific). Cellular TAG and cholesterol

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concentrations were normalized by total protein and displayed as µg/mg protein.

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Measurement of fatty acids in SCD1 knockout GMEC. The SCD1 knockout

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GMEC and control group were cultured in 60mm culture dishes until 90% confluence

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and the cells were washed with 1 mL PBS buffer for three times. Total fatty acid was

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extracted using 2 mL aliquot of 2.5% (vol/vol) sulfuric acid :methanol and then

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transferred to a 10 mL glass tube for methyl esterification as previously described46.

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Methylated lipid samples were analyzed by gas chromatography (Agilent 7890A;

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Agilent Technologies Inc., Santa Clara, CA) using a 100 meters HP-5 column

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(Agilent Technologies Inc.) and the hydrogen flame ionization detector as previously

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described48. Relative proportions of C16:0, C16:1, C18:0 and C18:1 in GMEC were

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determined as percentage of the total peak area that could be identified48. The

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desaturation index was calculated as the ratio of unsaturated fatty acid to the sum of

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unsaturated and saturated fatty acids, as previously reported49.

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Statistical analysis. Statistical analysis for gene expression was performed using

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GLM procedure of SAS (v9.4) with cell type (knockout, wild type) as main effect and 13



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replicates as random. For the other parameters the statistical analysis was performed

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using SPSS 19.0 statistics software (SPSS, Inc., Chicago, IL) using Student’s t test.

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Significant differences between the groups were considered significant at *P < 0.05.

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RESULTS

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Cleavage efficiency detection and construction of HDR donor vector. After treated

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with different concentrations of puromycin, the minimal lethal dose was 1.0 µg/ml for

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GMEC (Suppl. Figure 1). This concentration was used for cell selection. Among three

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sgRNAs, sgRNA2 had no significant cleavage, whereas sgRNA1 and sgRNA3 had

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8.3% and 19.4% cleavage efficiency, respectively (Figure 1B). There were different

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types of nucleotide indels in these cell pools (Figure 1D). Based on above results, we

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selected the sgRNA3 for the subsequent experiment.

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CRISPR/Cas9 induced sequence modification at SCD1 gene locus by NHEJ and

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HDR repair pathways. For the NHEJ pathway, we obtained 42 single clones and

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only one clone was identified with a gene modification representing 24 nucleotides

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deletion and 1 nucleotide mutation in the SCD1 exon (Figure 1C and 1E). These

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results indicated the Cas9/sgRNA system induced double strand break in GMEC,

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causing DNA fragments indels.

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For the HDR pathway, among 50 single clones obtained, we detected two clones

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that had both the 5’ homology arm and 3’ homology arm insertion, and one single

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clone only had the 5’ homology arm insertion (Figure 2B). Among the two clones

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with both arms insertion, only for clone 10, that had a smaller insertion in the 5’

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compared to clone 6 (Figure 2B), cells survived and proliferated. The green 14


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fluorescence could be detected in all the cells under the fluorescent microscope with

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the clone 10 (Figure 2C) indicating successful insertion of GFP and puromycin coding

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section in the SCD1 gene locus. The sequence in the genome at this site was shown in

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supplementary material.

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The test primer for NHEJ pathway was used to examine if there was

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NHEJ-mediated genome editing in the allele in HDR pathway mediated knockout

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cells. PCR was performed by using primers for NHEJ and using the genome DNA

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extracted from HDR group cells as templates. Small DNA fragment were amplified.

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This indicated only one allele contained GFP and puromycin insertion. Through

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T7EN1 assay, no nucleotides mutation was detected in this allele that did not contain

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GFP and puromycin insertion (Suppl. Figure 2). These results suggested that the

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SCD1 gene locus could be repaired by HDR pathway and did not generate the indels

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caused by NHEJ repair pathway. Taken together, these two single clones constructed

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by NHEJ and HDR pathways had genome sequence modifications of SCD1 gene

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locus and they were selected for further analysis.

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The expression of SCD1 decreased in genome modified GMEC without off-target

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effect. For off-targets detection, ten off-target sites (Figure 3A) were chosen for

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examination by T7EN1 assay (Figure 3B and 3C). The cleavage bands were visible in

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OT3 and OT6 both in wild type and knockout cells. These results indicated that no

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off-targets were detected in knockout cells and there were nucleotide mutations in the

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genome of wild type cells at off-target site 3 and 6 (Suppl. Figure 3).

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The relative mRNA and protein expression were measured by RT-qPCR and 15



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Western blot. The mRNA level of SCD1 decreased by about 80% in both HDR-and

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NHEJ-mediated genome editing, and the protein level of SCD1 decreased about 50%

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(Figure 4A and 4B). These data indicated that the use of either HDR and NHEJ and

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selection of the viable clone resulted in monoallelic knockout of SCD1.

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Knockout of SCD1 affected TAG, cholesterol, and the desaturation index.

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Knockout of SCD1 in GMEC decreased significantly the content of cellular TAG and

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cholesterol (P < 0.05) (Figure 5). The 16:1 and C18:1 desaturation index was

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decreased significantly (P < 0.05) as consequence of the knockout of SCD1 in GMEC

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(Figure 6). The percentage of C16:0 was increased (P < 0.05) while the percentage of

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C16:1 cis7 (P < 0.05) and C18:1 cis9 (P < 0.01) were decreased in SCD1 knockout

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cells compared to wild type. However, there was no significant difference for C18:0

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between SCD1 knockout and wild type GMEC (Table 2).

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Knockout of SCD1 affected the expression of genes related to de novo fatty acid

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synthesis. To determine whether the knockout of SCD1 was the sole cause of the

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decrease in TAG, cholesterol and unsaturated fatty acids and if it affected the

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expression of other genes related to milk fat synthesis, we assessed the expression of

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several genes related to fatty acid metabolism. Compared with the wild type GMEC,

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knocking out SCD1 gene both using HDR or NHEJ pathway decreased the expression

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of fatty acid transporters FABP3 and FABP4, the key de novo fatty acid gene FASN,

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ACACA (with a tendency of P = 0.08 when HDR was used), and SREBF1 (Figure 7, 8

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and 9). The use of NHEJ pathway also decreased the expression of ELOVL5 but

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increased the expression of ELOVL6. The use of HDR pathway tended (P = 0.07) to 16


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decrease the transcription of FADS1. The transcription of DGAT2 was significantly

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increased when the HDR pathway was used but was only numerically increased when

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NEHJ pathway was used. No other genes were affected by the knockout of SCD1

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(Figure 7 and 8).

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DISCUSSION

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CRISPR/Cas9-mediated genome editing provides an easier and more efficient

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method to obtain gene modification in any type of cell11, 50 compared with other

328

genome editing systems, like zinc-finger nucleases51 and transcription activator-like

329

effector nucleases52. To our knowledge, this is the first study where a genome editing

330

was performed using CRISPR/Cas9 technology by both NHEJ and HDR pathways in

331

goat mammary cells. Using the NHEJ pathway we obtained monoallelic SCD1

332

knockout by the deletion of eight amino acids and one amino acid mutation, whereas

333

in the HDR pathway the selection markers were inserted into the SCD1 genomic

334

sequence and the presence of a stop codon in the puromycin-coding sequence induced

335

a transcriptional termination, shortening the mRNA of SCD1. Taken together, our

336

results demonstrated that both the NHEJ- and HDR-mediated gene editing can be

337

applied successfully in primary goat mammary gland epithelial cells.

338

RNAi has significant off-target effects and unpredictable knockdown efficiencies

339

53

340

CRISPR/Cas9 technology has the advantage of complete knockout for a certain gene.

341

RNAi requires endogenous factors to form RISC (RNA-induced silencing complex) 54

342

while CRISPR system uses the exogenous crRNA and tracrRNA together with Cas9

. When a residual expression is enough for the protein activity, compared to RNAi,

17



343

endonuclease. As a result, the efficiency of RNAi may be more dependent from the

344

status of the cell55. A previous report indicated that compared with shRNA-based

345

system and CRISPR-interference (CRISPRi), the CRISPR technology performed best

346

in identifying essential genes with less off-target effect and consistency in different

347

cell lines56. In ruminant mammary gland, CRISPR/Cas9 genome editing tool also

348

provides a more reliable method to study fatty acid metabolism. In our experiment the

349

expression of DGAT2 was increased significantly in SCD1 knockout GMEC. This is

350

contrary to what previously observed using siRNA method9. The differences on the

351

effect of DGAT2 between the two studies might be due to off-target effects usually

352

obtained using RNAi but not CRISPR/Cas9.

353

In the HDR-directed knockout method, three single clones were selected with

354

three different status. Bands corresponding to the 3’ and 5’ junctions displayed higher

355

intensity in clone 6 when compared to clone 10 likely due to a more complete biallelic

356

deletion of SCD1; the colony, however, could not be cultured for an extended period

357

of time and underwent apoptosis in the first few days. The likely biallelic deletion of

358

SCD1 could have affected the viability of the clone, considering that SCD1 is

359

essential for the de novo formation of monounsaturated fatty acids. Furthermore, an

360

increase of saturated fatty acids can be toxic when excessive. A possible explanation

361

for this phenomenon is the presence of a biallelic SCD1 knockout in clone 6. As

362

consequence of the complete SCD1 knockout the cells could have been deprived of

363

adequate amount of MUFA, an essential substrate for phospholipid synthesis, an

364

important component of the cell membrane57. Further, SFA accumulation can trigger 18


Page 18 of 47

Page 19 of 47


365

endoplasmic reticulum stress, which would ultimately lead to cell death58.

366

Interestingly, biallelic SCD1 knockout mice are viable59,

367

monogastrics, ruminants absorb from the diet little amount of unsaturated fatty acids,

368

owned to the presence of the large biohydrogenation of unsaturated fatty acids in the

369

rumen61. Thus, it is possible that cells in ruminants are more dependent from the

370

intracellular desaturation, mainly driven by the activity of SCD1, to obtain

371

unsaturated fatty acids to be inserted into the cellular membrane62.

60

. Different than

372

It is interesting to highlight the fact that knockdown of SCD1 using siRNA in

373

GMEC did not affect cell viability9. siRNA-mediated gene knockdown is a temporary

374

gene deficiency; thus, detrimental effect of decrease or lack of SCD1 might be not

375

visible due to the brief duration of the deficiency. In CRISPR/Cas9-mediated gene

376

knockout the gene deficiency is permanent and the detrimental effect has more likely

377

to be expressed phenotypically. This might explain the difference in cell viability

378

observed between likely biallelic deletion of SCD1 using CRISPR/Cas9 and SCD1

379

knockdown using siRNA in GMEC.

380

The efficiency of any CRISPR-mediated gene editing endeavor is limited by the

381

presence of unspecific mutagenesis, or off-target effects. Although complete

382

homology is required to achieve significant rates of mutation, the literature shows that

383

mutagenesis can be detected on sequences with up to five mismatches, particularly

384

when sequence homology is maintained 10-12 nucleotides upstream of the PAM63. In

385

our study, no off-targets were detected among the ten predicted by Cas-OFFinder.

386

Although the predicted off-target site 3 and 6 have cleavage bands, they are both the 19



Page 20 of 47

387

natural mutations of one allele as also observed in the wild type cells, thus they could

388

be recognized by T7 endonuclease.

389

Our results indicated that we successfully got SCD1 monoallelic knockout in

390

GMEC by either causing indels via the NHEJ pathway or inserting the selection

391

markers to disrupt the coding frame by HDR pathway. The disruption of SCD1 in its

392

second exon caused a 50% decrease of SCD1 protein level; mRNA expression,

393

however, was affected more dramatically. The 80% decreased in SCD1 transcript

394

observed

395

expression/activation of the transcription factor SREBP1 known to bind to the SCD1

396

promoter in goats9.

may

be

caused

by

the

significant

or

numerical

decrease

in

397

The decrease of TAG, cholesterol and fatty acid desaturation index in knockout

398

cells provided further support for a successfully knockout of SCD1; however, the

399

decrease of TAG and desaturase indexes was only approx. 25% in knockout vs. wild

400

type cells, which was similar between the clone obtained by NHEJ and the one by

401

HDR. The decrease of TAG was less than proportional compared to the decrease in

402

SCD1 protein expression, indicating that the effect on TAG synthesis was mostly

403

retained and compensated by the wild-type allele. The decrease in cholesterol was

404

more pronounced (≥40%) and with larger decrease in the clone obtained using NHEJ

405

compared to HDR. The former had a larger decrease in expression and activity of

406

SREBP1 compared to the latter, indicating that the decrease in cholesterol was partly

407

due to a decrease expression/activity of SREBP1.

408

The reduction of TAG in SCD1 knockout cells appeared to be a consequence of a 20


Page 21 of 47


409

reduced de novo fatty acid synthesis and intracellular LCFA transport. This is

410

indicated by an overall decrease in expression of FASN, SREBF1, FABP3, and FABP4.

411

The acetyl-CoA carboxylase was also affected by SCD1 knockout. Its expression was

412

decreased, although significant only with NHEJ, but also its activity was likely

413

inhibited by an increased proportion of C16:064 with the consequent reduction of

414

malonyl-CoA. The latter is used by FASN to synthesize long chain fatty acid65, 66.

415

It is unclear how a deficiency of SCD1 would affect the expression of other

416

lipogenic genes in our study. However, SCD1 appears to regulate SREBP1. In Scd1

417

knockout mice, the expression of Srebf1 and Fasn were both downregulated67, similar

418

to our findings. A very similar results was obtained in mice with liver specific Scd1

419

knockout where a decreased expression of Srebf1c and reduced lipogenesis were

420

detected28. SREBP1 has a central role in the regulation of milk fat synthesis-related

421

genes in all species, including goat46. As for other species, FASN is among the genes

422

regulated by SREBP1 also in goat68; thus, the decreased expression of FASN in SCD1

423

knockout cells is likely consequence of the decreased expression/activity of SREBP1.

424

An important role of SCD1 in the activity of SREBP1 was also revealed in Scd1

425

knockout mice fed fructose where the up-regulation of Srebf1 was observed only

426

when supplemented with oleic acid, the main product of SCD1, but not with palmitate

427

or stearate26. No data in this regard are available for goats or other ruminants;

428

however, oleic acid does not seem to have any transcriptional effect on SCD1 or

429

SREBP1 down-stream genes in bovine69. Furthermore, activation of SREBP1 in

430

Drosophila was inhibited by palmitic acid, which is the substrate of SCD170. In cancer 21



431

cells, high SCD1 activity promotes an increase in MUFA-to-SFA ratios in cell

432

membrane activating tyrosine kinase receptors and their membrane-interacting

433

signaling mediators such as PI3K, Akt and Ras. These signaling mediators affect the

434

activity of mTOR that is known to regulate SREBP158. All the above studies about the

435

effect of SCD1 on SREBP1 where performed in non-ruminant species. Despite

436

important differences with ruminants, for instance the absence or minor effect on

437

transcription of genes by oleate69, it appears likely that the same effect of SCD1 on

438

SREBP1 was present in our cells.

439

ELOVL5, ELOVL6 and SCD1 jointly participate in fatty acid de novo synthesis.

440

Previous studies in GMEC indicated both ELOVL5 and ELOVL6 do not influence

441

SCD1 expression72, 73. Knockout of SCD1 in NHEJ pathway decreased the content of

442

C16:1, the substrate of ELOVL5, and increased C16:0, the substrate of ELOVL6. The

443

change of gene expression may due to their substrates alteration. The different

444

expression of ELOVL5 and ELOVL6 in NHEJ and HDR pathways knockout GMEC

445

may be caused by the cell models obtained from different ways. The different DNA

446

repair pathways would have influence on some genes expression, but the mechanism

447

is still not clear.

448

In bovine mammary gland, FABP3 likely plays an essential role in providing

449

fatty acids to SCD1 while FABP4 takes the unsaturated fatty acid coming from SCD1

450

to transport it to the enzymes involved in TAG synthesis8. It is interesting in our study

451

that the transcription of both fatty acid transporters was down-regulated by knocking

452

out SCD1. It is unclear what the physical connections between the downregulation of 22


Page 22 of 47

Page 23 of 47


453

the fatty acid binding proteins and SCD1, however it makes biological sense

454

considering the concerted function of the two FABP with SCD1 for the synthesis of

455

TAG.

456

In conclusion, we successfully obtained SCD1 monoallelic knockout GMEC by

457

CRISPR/Cas9-mediated gene editing both through NHEJ and HDR pathways. Our

458

data indicated that full SCD1 knockout is likely deleterious for GMEC. The

459

monoallelic knockout of SCD1 decreased the amount of TAG, cholesterol, and

460

desaturase index and negatively affected the expression of several key genes related to

461

de novo fatty acid synthesis. Using a more effective knockout system compared to

462

RNAi, our data confirm SCD1 being an important enzyme in synthesis of TAG and

463

unsaturated fatty acids in goat milk. It remains to be determined the physical

464

relationship between SCD1 and several of the genes which expression was decreased

465

by deleting SCD1, chiefly SREBF1 being a key transcription factor in milk fat

466

synthesis.

467 468 469

ACKNOWLEDGEMENTS This research was supported by National Natural Science Foundation of China (Beijing, China; 31772575).

470

23



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58. Igal, R. A., Stearoyl CoA desaturase-1: New insights into a central regulator of

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59. Dobrzyn, P.; Dobrzyn, A.; Miyazaki, M.; Cohen, P.; Asilmaz, E.; Hardie, D. G.;

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Friedman, J. M.; Ntambi, J. M., Stearoyl-CoA desaturase 1 deficiency increases fatty

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acid oxidation by activating AMP-activated protein kinase in liver. Proc Natl Acad Sci

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U S A 2004, 101, 6409-14.

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60. Miyazaki, M.; Man, W. C.; Ntambi, J. M., Targeted disruption of stearoyl-CoA

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desaturase1 gene in mice causes atrophy of sebaceous and meibomian glands and

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depletion of wax esters in the eyelid. J Nutr 2001, 131, 2260-2268.

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61. Jenkins, T. C.; Wallace, R. J.; Moate, P. J.; Mosley, E. E., Board-invited review:

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Recent advances in biohydrogenation of unsaturated fatty acids within the rumen

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microbial ecosystem. J Anim Sci 2008, 86, 397-412.

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62. Ntambi, J. M., Stearoyl-CoA Desaturase Genes in Lipid Metabolism. 2013.

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63. Kuscu, C.; Arslan, S.; Singh, R.; Thorpe, J.; Adli, M., Genome-wide analysis

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reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat

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Biotechnol 2014, 32, 677-83.

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64. Paton, C. M.; Ntambi, J. M., Biochemical and physiological function of

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stearoyl-CoA desaturase. Am J Physiol Endocrinol Metab 2009, 297, 28-37.

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65. Jenni, S.; Leibundgut, M.; Boehringer, D.; Frick, C.; Mikolásek, B.; Ban, N.,

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Structure of Fungal Fatty Acid Synthase and Implications for Iterative Substrate

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Shuttling. Science 2007, 316, 254.

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66. Livore, V. I.; Tripodi, K. E.; Uttaro, A. D., Elongation of polyunsaturated fatty

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acids in trypanosomatids. FEBS J 2010, 274, 264-274.

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67. Ntambi, J. M.; Miyazaki, M.; Stoehr, J. P.; Lan, H.; Kendziorski, C. M.; Yandell, 32


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B. S.; Song, Y.; Cohen, P.; Friedman, J. M.; Attie, A. D., Loss of Stearoyl-CoA

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Desaturase-1 Function Protects Mice against Adiposity. Proc Natl Acad Sci U S A

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2002, 99, 11482-6.

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68. Li, J.; Luo, J.; Xu, H.; Wang, M.; Zhu, J.; Shi, H.; Haile, A. B.; Wang, H.; Sun, Y.,

673

Fatty acid synthase promoter: characterization, and transcriptional regulation by sterol

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regulatory element binding protein-1 in goat mammary epithelial cells. Gene 2015,

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561, 157-64.

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69. Bionaz, M.; Osorio, J.; Loor, J. J., TRIENNIAL LACTATION SYMPOSIUM:

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Nutrigenomics in dairy cows: Nutrients, transcription factors, and techniques. J Anim

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Sci 2015, 93, 5531-5553.

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70. Seegmiller, A. C.; Dobrosotskaya, I.; Goldstein, J. L.; Ho, Y. K.; Brown, M. S.;

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Rawson, R. B., The SREBP Pathway in : Regulation by Palmitate, Not Sterols. Dev

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71. Shi, H. B.; Du, Y.; Zhang, C. H.; Sun, C.; He, Y. L.; Wu, Y. H.; Liu, J. X.; Luo, J.;

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Loor, J. J., Fatty acid elongase 5 (ELOVL5) alters the synthesis of long-chain

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unsaturated fatty acids in goat mammary epithelial cells. J Dairy Sci 2018, 101,

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72. Shi, H. B.; Wu, M.; Zhu, J. J.; Zhang, C. H.; Yao, D. W.; Luo, J.; Loor, J. J., Fatty

687

acid elongase 6 plays a role in the synthesis of long-chain fatty acids in goat

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689 690 33



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691

Table 1. Test primers for NHEJ and 5’ and 3’ homology arms for HDR pathway

692

induced SCD1 knockout. Primer Test primer for NHEJ

Sequence (5’ to 3’) F: GGGAGTGAAGTGGTCCCTAC

Size (bp) 583

R: CAGCCCCAACACCGAAATTA 5’ homology arm clone primer

5-F: AGCACCCCATACCCAAGACT

1193

5-R: GGATAAGGAGGGCCCAAAGC 3’ homology arm clone primer

3-F: TCATCTCTCATTTCAGGGCG

1063

3-R: TGTGTCTGCAGCATCCAGTT 5’ junction primer

5j F: AATAAGAGCCCTTCCTGGTTT

1697

5j R: GGTTCTACGTTAGTGGGAGTTT 3’ junction primer

3j F: TCATAATCAGCCATACCACA 3j R: CCTAGTGCCCATCCATTT

693 694

34


1698

Page 35 of 47


695

Table 2. Effects of SCD1 monoallelic knockout on fatty acid composition in GMEC. Fatty acid

HDR-CTR

HDR-KO

Fatty acid

NHEJ-CTR

NHEJ-KO

C16:0(%)

23.19a±0.15

26.73b±0.70

C16:0(%)

23.41A±0.49

25.38B±0.23

C16:1(%)

2.15a±0.03

1.90b±0.00

C16:1(%)

1.97a±0.09

1.69b±0.00

C18:0(%)

17.34±0.15

16.80±0.20

C18:0(%)

17.34±0.15

17.31±0.33

C18:1 cis9(%)

20.56a±0.62

18.66b±0.15

C18:1 cis9(%)

20.10a±1.27

16.98b±0.14

696

Fatty acid data are reported as proportion of the total fatty acids. Statistical

697

significance between knockout (KO) and wild type GMEC (CTR) was as follow:

698

lower case letters, P < 0.05; upper case letters, P < 0.01. Data are presented as means

699

± SEM.

700

35



701

Figure legends

702

Figure 1. sgRNAs target sites selection of SCD1 gene in Capra hircus genome and

703

NHEJ-mediated gene modification. (A) SCD1 gene locus and sgRNAs design. PAM

704

motifs are bold and underlined. (B) Cleavage efficiency of Cas9/sgRNA at three target

705

sites. CTR denotes the DNA isolated from GMEC transfected with pSpCas9

706

(BB)-2A-Puro without sgRNA sequence. The cleavage efficiency was quantified with

707

T7EN1 cleavage assay and analyzed by ImageJ. (C) Cleavage efficiency of the

708

GMEC single clone through NHEJ-mediated SCD1 gene modification by T7EN1

709

assay. (D) The sequences of modified SCD1 alleles. sgRNA target sequences are in

710

red text; PAM motifs are in green text bold and underlined; mutations are red and

711

lowercase; deletions (-), mutations (m) or wild type (WT) are shown to the right of

712

each sequence. (E) The sequence of the single clone detected in picture (C).

713

Figure 2. Insertion and selection of the SCD1 transgenic single clone by

714

HDR-mediated genome editing. (A) Schematic representation of the HDR donor

715

vector and 5’ or 3’ junction PCR primers position. EGFP: enhanced green

716

fluorescence protein; P2A: porcine teschovirus-1 2A; PuroR: puromycin resistance

717

selection marker; 5j F: 5’ junction PCR forward primer; 5j R: 5’ junction PCR reverse

718

primer; 3j F: 3’ junction PCR forward primer; 3j R: 3’ junction PCR reverse primer. 5j

719

F and 3j R were the primers for the regions outsides the homologous arms in the

720

genome, and 5j R and 3j F were primers for the donor vector region. (B) 5’ and 3’

721

junction PCR for the GMEC single clones detection which were transfected with both

722

sgRNA/Cas9 vector and donor vector. (C) Photograph of the single clone 10 GMEC

723

(KO) and a wild type GMEC counterpart (CTR). Scale bar is 100 µm.

724

Figure 3. Off-target sites of sgRNA3 in NHEJ- and HDR-mediated SCD1 knockout

725

GMEC. The sgRNA sequences end with PAM motif. (A) 10 off-target sites predicted

726

by online website were selected. The off-target nucleotide bases matching with the

727

sgRNA and the PAM sequence are highlighted in orange and red respectively. (B)

728

Off-target sites detection in NHEJ-mediated knockout single clone and (C)

729

HDR-mediated knockout single clone. Top pictures in B and C are PCR products and 36


Page 36 of 47

Page 37 of 47


730

bottom pictures are the results of T7EN1 cleavage assay. OT: off-target, WT: wild

731

type for each off-target site in control group.

732

Figure 4. SCD1 knockout in GMEC both through HDR and NHEJ pathway.

733

Real-time quantitative PCR analysis and protein level of SCD1 in NHEJ (A) and

734

HDR (B) knockout GMEC. Values are mean ± SEM for three independent

735

experiments. *P < 0.05 v. control.

736

Figure 5. Monoallelic knockout of SCD1 decreased the content of cellular

737

triacylglycerol (TAG) and cholesterol. Quadrants A and C report the amount of

738

triacylglycerol (TAG) and quadrants B and D the amount of cholesterol in NHEJ and

739

HDR groups. Values are presented as mean ± SEM for three independent experiments.

740

*P < 0.05 v. control.

741

Figure 6. Effects of SCD1 knockout on fatty acid desaturation index. Desaturation

742

index of C16:1 (A and C) and C18:1 (B and D) in NHEJ and HDR pathway SCD1

743

monoallelic knockout compared with wild type GMEC. Values are mean ± SEM for

744

three independent experiments. *P < 0.05 v. control.

745

Figure 7. Monoallelic knockout of SCD1 through NHEJ pathway affects mRNA

746

expression level of genes related to milk fat synthesis. Values are mean ± SEM for

747


748

Figure 8. Monoallelic knockout of SCD1 through HDR pathway affected mRNA

749

expression level of genes related to milk fat synthesis. Values are mean ± SEM for

750


751

Figure 9. Relative mRNA expression and protein level of precursor SREBP1 and

752

nuclear SREBP1 (mature SREBP1) in NHEJ-KO and HDR-KO groups. Values are

753

mean ± SEM. *P < 0.05 v. control.

754

37



755 756

Figures Figure 1.

757 758

38


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Page 39 of 47


759

Figure 2.

760 761

39



762

Figure 3.

763 764

40


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Page 41 of 47


765

Figure 4.

766 767

41



768

Figure 5.

769 770

42


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Page 43 of 47


771

Figure 6.

772 773

43



774

Figure 7.

775 776

44


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Page 45 of 47


777

Figure 8.

778 779

45



780

Figure 9.

781 782

46


Page 46 of 47

Page 47 of 47


783

TOC graphic

784 785

Legends: This study aims at investigating the function of SCD1 gene using

786

CRISPR/Cas9 technology in goat mammary epithelial cells. Firstly, non-homology

787

end joining (NHEJ) and homology directed repair (HDR) pathway were both used to

788

get the SCD1 monoallelic knockout cells. Then the content of cellular triacylglycerol,

789

cholesterol, fatty acid and fatty acid metabolism gene expression were measured. Our

790

results confirmed the important role of SCD1 on milk fat metabolism in goat

791

mammary cells.

792

47


Cas9-mediated Stearoyl-CoA desaturase 1 (SCD1

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