Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference

. 2022 Dec 24;8(1):148-156.


doi: 10.1016/j.synbio.2022.12.005.


eCollection 2023 Mar.

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Rochelle Carla Joseph et al.


Synth Syst Biotechnol.


.

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Abstract

The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.


Keywords:

CRISPR Cas12a; CRISPRi; Clostridium.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures


Fig. 1



Fig. 1

Distribution of TTN, TTTV and NGG PAM sites in (A) Clostridium acetobutylicym and (B) Clostridium pasteurianum: Graph shows distribution of PAM consensus sequences within 500 bp of each other. PAM site data was obtained using a python script designed to scan genomes and determine the number of and distance between sequential occurrences of the consensus sequence for respective Cas proteins.


Fig. 2



Fig. 2

Repression of spo0A using dFnCas12a in Clostridium acetobutylicum: (A) Transcriptional changes in spo0A, adc, adhE1, ctfA and ctfB following CRISPR-based repression of spo0A. Samples were obtained 24 h after induction of dFnCas12a with 10 mM lactose for RT-qPCR analysis. dFnCas12a targets the antisense strand, upstream of transcriptional start site between the sigma A/sigma K binding sites (1) and sigma H binding site/0A box (2) using g39 as shown. Lines are indicative of mean relative expression. (B) Growth, (C) pH and (DG) metabolite production data over a 96-h fermentation following dFnCas12a induction at t = 0. All data are representative of biological duplicates. pJRJ001-g58, harboring a non-complementary gRNA sequence, is used as the no target control. p value summary: *p < 0.05, **p < 0.01.


Fig. 3



Fig. 3

Simultaneous repression of pdc and adc expression in Clostridium acetobutylicum using a single CRISPR array: (A) Map of synthetic CRISPR array (B) Transcriptional changes in adc and pdc following simultaneous repression of the two genes via a synthetic CRISPR array (g76). Samples were obtained 24 h after induction of dFnCas12a with 10 mM lactose for RT-qPCR analysis. Lines indicate the mean relative expression. (C) Concentration of acids and solvents produced when pdc (g73) and adc (g74) are targeted for repression individually, and simultaneously via a synthetic CRISPR array. Samples were obtained 72 h following dFnCas12a induction at t = 0. All data are representative of biological triplicates. Error bars indicate standard deviation. pJRJ001-g58 is used as the no target control. p value summary: *p < 0.05, **p < 0.01.


Fig. 4



Fig. 4

Repression of hydA using dFnCas12a in Clostridium pasteurianum: (A) Map showing gRNA target region (B) Transcriptional changes in hydA (g58) following CRISPR-based repression. Samples were obtained 48-h after induction of dFnCas12a with 10 mM lactose for RT-qPCR analysis. Data are representative of biological duplicates. Error bars indicate standard deviation. pJRJ001-g1, harboring a non-complementary gRNA sequence, is used as the no target control p value summary: *p < 0.05, **p < 0.01. (C) Glycerol consumption and metabolite production 72 h after dFnCas12a induction. Data are representative of triplicates. No significant change in metabolite profile was observed.


Fig. 5



Fig. 5

Gene repression of dha operon in Clostridium pasteurianum following CRISPR-based repression of dhaB and dhaT: (A) gRNAs were designed to target sense and antisense strands of dhaB and dhaT gene respectively (B) 1,3-Propanediol concentrations following dFnCas12a expression. Samples were obtained 4 h after induction of dFnCas12a expression with 10 mM lactose. Lactose was added to media at mid-exponential phase (t = 4 h). control p value summary: *p < 0.05, **p < 0.01. (C) Concentrations of glycerol and metabolites produced over a 72-h fermentation of cells harboring pJRJ001-g26 following induction of dFnCas12a at t = 0 with 10 mM lactose. No significant change in metabolite profile was observed. All data are representative of triplicates. Error bars indicate standard deviation. pJRJ001-g52 is used as the no target control.

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