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Technical Note

In-yeast engineering of a bacterial genome using CRISPR/Cas9 Iason TSARMPOPOULOS, Géraldine GOURGUES, Alain Blanchard, Sanjay VASHEE, Joerg JORES, Carole LARTIGUE, and Pascal SIRAND-PUGNET ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00196 • Publication Date (Web): 23 Nov 2015 Downloaded from http://pubs.acs.org on November 26, 2015

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ACS Synthetic Biology

TITLE: In-yeast engineering of a bacterial genome using CRISPR/Cas9

PAPER TYPE: Technical note

Authors and affiliation: Iason Tsarmpopoulos1,2, Géraldine Gourgues1,2, Alain Blanchard1,2, Sanjay Vashee3, Joerg Jores4, 5, Carole Lartigue1,2 and Pascal Sirand-Pugnet1,2* 1

INRA, UMR 1332 de Biologie du Fruit et Pathologie, F-33140 Villenave d’Ornon, France

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Univ.

Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, F-33140 Villenave d’Ornon, 3 The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, 20850 MD USA,

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International Livestock Research

Institute (ILRI), PO Box 30709, 00100 Nairobi, Kenya, 5 Institute of Veterinary Bacteriology, University of Bern, Laenggass-Str. 122, CH-3001 Bern, Switzerland

*Corresponding author: Pascal Sirand-Pugnet, [email protected]

ABSTRACT One remarkable achievement in synthetic biology was the re-construction of mycoplasma genomes and their cloning in yeast where they can be modified using available genetic tools. Recently, CRISPR/Cas9 editing tools were developed for yeast mutagenesis. Here, we report their adaptation for the engineering of bacterial genomes cloned in yeast. A seamless deletion of the mycoplasma glycerol-3phosphate oxidase-encoding gene (glpO) was achieved without selection in one step, using 90 nt paired oligonucleotides as templates to drive recombination. Screening of the resulting clones revealed that more than 20% contained the desired deletion. After manipulation, the overall integrity of the cloned mycoplasma genome was verified by multiplex PCR and PFGE. Finally, the edited genome was backtransplanted into a mycoplasma recipient cell. In accordance with the deletion of glpO, the mutant mycoplasma was affected in the production of H2O2. This work paves the way to high-throughput manipulation of natural or synthetic genomes in yeast.

KEYWORDS: genome engineering, Mycoplasma, Saccharomyces cerevisiae, CRISPR/Cas9, genome transplantation, seamless gene deletion

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INTRODUCTION Recent cloning and assembly of a synthetic bacterial genome in yeast opened the way to large scale genome engineering using genetic tools available in yeast1–3. In these approaches, yeast is the workshop to introduce all kinds of modifications in the cloned genome. This potentiality has raised high hopes for the functional genomics of bacteria for which no efficient genetic tools are available, such as mycoplasmas. A significant variety of methods, including TREC and TREC-IN, have already been successfully used to delete or modify specific genes of a mycoplasma genome cloned in yeast4,5. However, while effective, these methods remain labor-intensive and more efficient approaches are desirable to accelerate genome engineering. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. From the natural system identified in Streptococcus pyogenes, genetic tools have been derived for genome engineering in prokaryotes and eukaryotes 6. Recently, CRISPR/Cas9 tools have been adapted to yeast. Using a two-plasmid system, DiCarlo et al. reported targeted gene mutagenesis in S. cerevisiae with efficiency rates close to 100%7. We describe here, the adaptation of this CRISPR/Cas9 tool for an efficient one-step seamless deletion of a gene in a mycoplasma genome cloned in yeast.

RESULTS AND DISCUSSION Construction of a CRISPR/Cas9 workshop for mycoplasma genome engineering The goal of this study was to develop and evaluate the efficiency of CRISPR/Cas9 tool for fast and efficient seamless deletions of genes in a mycoplasma genome cloned in yeast. In a first step, we transformed a yeast strain that contained a complete mycoplasma genome with a plasmid constitutively expressing Cas9. We used a W303a yeast strain harboring the 1.2 Mbp complete genome of Mycoplasma mycoides subsp. capri GM12 (Mmc) maintained as a circular extra-chromosome after integration of yeast replication elements ARSH4 and CEN6 and the selection marker HIS31,8, henceforth termed W303a/Mmc. The plasmid p414-TEF1p-Cas9-CYC1t7 designed for the constitutive expression of a eukaryotic codonoptimized version of Cas9 was transformed into W303a/Mmc and transformants were isolated on selective medium. Transformed yeast W303a/Mmc/p414-TEF1p-Cas9-CYC1t was grown in selective liquid medium and stored at -80°C. Customization of a plasmid for expression of gRNA and integration of glpO target sequence The second step was to customize the original plasmid p426-SNR52p-gRNA. CAN1.Y-SUP4t7 from Dicarlo et al. that contained an expression cassette for a chimeric guide RNA (gRNA) including a 20 nt spacer specific to the target in fusion with the structural component (tracrRNA). Using the Gibson assembly

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method9, the original spacer targeting yeast gene CAN1 was replaced by a cloning spacer that includes two sites for the type IIS restriction enzyme AarI, in reverse and opposite direction (Figure 1). In the resulting pgRNA.AarI plasmid, the cloning spacer can be easily replaced by direct insertion of annealed oligonucleotides with compatible overhangs after Aar1 digestion, as described by Ran and co-workers10. The AarI cloning spacer was then replaced by annealed primers targeting the glpO gene from Mmc (MMCAP2_0219) to obtain the pgRNA.ΔglpO plasmid. The glpO gene encodes a glycerol-3-phosphate oxidase involved in the production of hydrogen peroxide and is considered as a virulence factor in several pathogenic mycoplasmas11–13. This gene is present in one copy in the Mmc genome. Design of the 20 bp spacer was performed taking into account the following criteria established by others14: (i) presence of a NGG consensus Protospacer Adjacent Motif (PAM) immediately downstream the spacer (in this case, TGG), (ii) G+C content between 20 and 80% (23.1%), (iii) absence of polyT (more than 4 T) that could stop transcription by type III RNA polymerase. Among these criteria, the G+C content is particularly relevant as it ranges from 23 to 40% in Mycoplasma species, with 23.9% for Mmc. The selected spacer targets the 1,164 bp glpO gene at position 726-745 (Figure 1). Once the CRISPR/Cas9 system has cleaved the targeted DNA, the double-strand break can be resolved either by error-prone non-homologous end joining or by homologous recombination when a DNA template is available. To take advantage of the highly efficient homologous recombination process in S. cerevisiae, we designed a 90 bp recombination template made of two annealed oligonucleotides, with arms of 45 bp that are complementary to the genome sequences immediately adjacent to the glpO coding sequence (Figure 1). Recombination with this synthetic template should result in the seamless deletion of glpO from the mycoplasma genome. Deletion of the glpO gene in the Mmc genome resident in yeast Yeast W303a/Mmc/p414-TEF1p-Cas9-CYC1t was co-transformed with the pgRNA.ΔglpO plasmid and the glpO recombination template. After selection of transformants, DNA extraction was performed on 12 pools of 20 colonies each followed by a PCR screening with primers located on both sides of the glpO gene (Figure 2). Eight pools tested showed a 483 bp amplification product corresponding to ΔglpO mutants, in addition to the 1,640 bp amplification product corresponding to the wild-type genomic structure. Two positive pools were selected and individual clones were screened to isolate mutants using the same process. A clear single 483 bp amplicon indicating the absence of the glpO gene was observed for 4 individual clones each within pools P7 and P8. Mixed profiles were observed for several colonies possibly due to the presence of both modified and unmodified copies of the Mmc genome in yeast cells. Similar results were obtained in two replicates of the experiment. The seamless deletion of glpO was

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confirmed in three clones by sequencing of the PCR products, indicating that homologous recombination had occurred as expected. Although our results confirmed the effectiveness of the CRISPR/Cas9 system developed by DiCarlo et al.7, but our rate of recombination was lower than the 100% (obtained when targeting the CAN1 gene of yeast). However, in this previous study, the targeted mutation was limited to the replacement of a GGC triplet by a TAG sequence to introduce a STOP codon and to delete the PAM sequence. In our study, we succeeded in the deletion of a 1,164 bp gene without selection, showing that CRISPR/Cas9 tools associated with the highly efficient homologous recombination capacity of yeast can be used for the deletion of a full gene within a cloned bacterial genome. Evaluation of mycoplasma genome integrity and back transplantation One of the described drawbacks of CRISPR/Cas9 tools is the problem of potential background off-target cleavage by Cas915. Despite a careful design of the gRNA, it is possible that during CRISPR/Cas9 manipulation, non-targeted cleavage of the mycoplasma genome occurs and results in the instability of the genome. Thus, the overall integrity of the mycoplasma genome resulting from the Cas9 based deletion of glpO was first evaluated in 4 clones using multiplex PCR that targeted 9 independent regions distributed across the Mmc genome [Table S1]. Two clones (P7.14 and P8.20) presented the 9 expected amplified fragments while several bands were missing for the two others, suggesting that genome alterations had occurred in the latter clones. In addition, PFGE analysis performed after mycoplasma chromosome linearization by PspXI restriction enzyme suggesting the global genome integrity of clones P7.14 and P8.20 with single band profiles that was identical to the wt control. In accordance with multiplex PCR profiles, a band of about 650 kbp was observed for the two other clones, indicating large deletions (Figure 2). Finally, back transplantation of the ΔglpO Mmc genomes from yeast clones P7.14 and P8.20 into a mycoplasma recipient cell was performed as previously described8. Transplants were obtained after five days for both yeast clones, demonstrating the viability of the resulting recombinants. A final PCR test was conducted on three clones to confirm that the glpO gene was actually deleted in the transplanted mycoplasmas. All three showed the expected PCR product, demonstrating the deletion of the glpO gene. The impact of glpO deletion on the production of H2O2 in presence of glycerol was investigated as described previously11. A concentration of 5-10 mg/L-1 of H2O2 was measured after a 100 min incubation of wt Mmc with 100 µM glycerol. In contrast, no trace of H2O2 production was detected for the three clones where the glpO gene had been deleted. This result was in complete accordance with previous studies11,16 showing that glpO is directly responsible for the release of highly cytotoxic H2O2 by mycoplasmas.

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CONCLUSION Here, we show that CRISPR/Cas9 systems can be used as efficient tool for the deletion of genes in bacterial genomes cloned in yeast. Other methods, such as TREC, have already been described to generate seamless deletions4. The TREC method was shown to be very efficient with up to 100% clones containing the correct modification at the end of the selection process. In comparison, the CRISPR/Cas9 method is currently less efficient with 20% clones having the correct modification. Both TREC and CRISPR/Cas9-based methods are highly accurate with all clones sequenced having the exact expected deletion. Given this, the lower level of efficiency in the CRISPR/Cas9-based method is more than acceptable since it is significantly faster to obtain the desired modification than the TREC method. Indeed, the TREC method is based on a two-step process that includes first, integration of a URA3 selectable marker at the targeted locus by homologous recombination followed by induction of a rarecutter endonuclease (I-SceI) to introduce a double-strand break close to the marker and a final homologous recombination event to delete the marker with a counter-selection step on 5-fluorouracil containing media. This time-consuming process is needed because the efficiency of homologous recombination in yeast is high but not enough to proceed without the initial step of using a selectable marker. According to our experience, one mutant can be generated in yeast using the TREC system in 4 weeks whereas 2 weeks are sufficient with the CRISPR/Cas9 system. In addition, the CRISPR/Cas9 method can be performed without selection potentially enabling iterative deletions of multiple genes without the need to remove selection markers at each cycle. While further developments of the method are currently undergoing to improve the efficiency and thereby accelerate the production of mutants with multi-target gRNAs, our work paves the way for high-throughput and reduced-cost manipulation of natural or synthetic genomes in yeast. Moreover, this work will foster the development of CRISPR/Cas9 based editing tools that can be directly applied to Mycoplasma.

MATERIAL AND METHODS Yeast and bacterial strains. The S. cerevisiae strain W303a-Mmc.cl1.1 clone 5 harboring the genome of Mmc GM128 was used for the evaluation of CRISPR/Cas9 tools. For back transplantation, restriction free Mycoplasma capricolum subsp. capricolum California Kid strain cl.17.5 (Mcap∆RE) was used as a recipient cell as described previously8. Plasmid constructions were achieved in Escherichia coli strain DH10B. Plasmids. Plasmids p414-TEF1p-Cas9-CYC1t and p426-SNR52p-gRNA.CAN1.Y-SUP4t developed by DiCarlo et al7 were acquired from the Addgene repository (references: #43802 and #43803, respectively). The p426-SNR52p-gRNA is a high-copy 2µ plasmid with URA3 selection marker whereas the p414-TEF1p-

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Cas9-CYC1t is a centromeric plasmid with a CEN6/ARSH4 origin and TRP1 as a selection marker. In the plasmid p414-TEF1p-Cas9-CYC1t, the Cas9 encoding gene is a codon-optimized version originally designed for expression in human cells17. Constitutive expression is controlled by a TEF1p promoter and nuclear localization is driven by a C-terminal SV40 tag. Expression of the gRNA from the p426-SNR52pgRNA.CAN1.Y-SUP4t and derived plasmids is under the control of the SNR52 promoter with the SUP4 3’ flanking sequence as a terminator. Replacement of the CAN1.Y 20 bp spacer by an AarI cloning spacer in p426-SNR52p-gRNA.CAN1.Y-SUP4t

was

achieved

by

Gibson

assembly9.

Using

p426-SNR52p-

gRNA.CAN1.Y-SUP4t as template, two overlapping PCR fragments containing the AarI cloning spacer were produced with primer pairs AarI_gRNA_modF/p426R and AarI_gRNA_modR/p426F (Table S1). Gibson assembly was performed and plasmid sequence was verified by sequencing. Integration of the glpO specific spacer was then conducted following the protocol of Ran et al10 with modifications. The enzymatic digestion of the pgRNA.AarI plasmid with AarI (ThermoScientific) was performed according to the manufacturer’s recommendations for 5h at 37°C to ensure full digestion of the plasmid. Equimolar amounts of complementary oligonucleotides with overhangs glpO_AarI_comp_F and glpO_AarI_comp_R (Table S1) were annealed by an initial denaturing step of 5 min at 95°C and a controlled cooling to 16°C with a ramp of 0.1°C per second. Ligation of the annealed oligonucleotides was conducted at 16°C overnight using the T4 ligase of Promega, with a 1:3 vector/insert ratio according to the manufacturer’s protocol. Ligation products were transformed into E. coli DH10B cells and recombinant clones were screened for the absence of an AarI site. Sequence of the pgRNA.glpO plasmid was finally verified by sequencing. Yeast transformation. Yeast cells were transformed using the standard lithium acetate protocol18. Approximately 200 ng of plasmid p414-TEF1p-Cas9-CYC1t were used to transform yeast strain W303a/Mmc to obtain yeast strain W303a/Mmc/p414-TEF1p-Cas9-CYC1t. For deletion of the glpO gene, the yeast strain W303a/Mmc/p414-TEF1p-Cas9-CYC1t was transformed with 200 ng of plasmid pgRNA.glpO and 1 nmol of annealed oligonucleotides Del_glpO and Del_glpOcomp as template for homologous recombination. After transformation, cells were incubated at 30°C for 1h in rich medium (YPDA) in agitation, washed with water, resuspended in selective medium and incubated at 30°C for 48 hours before plating on selective medium. Screening for mycoplasma genome modification, verification of mycoplasma genome integrity and back transplantation. Total genomic DNA was extracted from transformed yeast as described by Kouprina and Larionov19. PCR screening of glpO-deleted clones was conducted on pools of 20 clones and individual clones using primers glpODel_verif_F and glpODel_verif_R. The integrity of mycoplasma genome was first verified by multiplex PCR using 9 primer pairs distributed all over the chromosome (Table S1) and

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the Qiagen Multiplex PCR kit. PFGE analysis was conducted after in-plugs digestion of the mycoplasma genome by PspXI as previously described8. Back transplantation of the glpO-deleted Mmc genome into Mcap∆RE was performed as described previously8, except that the recipient cells were cultured at 30°C in SOB(+) medium. Quantification of H2O2 production. Release of H2O2 in the presence of glycerol by Mmc and mutant was determined using the MQuantTM peroxide test (Merck Millipore) as described by others11. Briefly, Mmc and ΔglpO Mmc were grown to mid-log phase. Cells were harvested and resuspended in HEPES buffer (HEPES 67.7 mM, NaCl 140 mM, MgCl2 7 mM, pH=7.3) and incubated for 1h at 37°C. Glycerol was added at a final concentration of 100 µM and H2O2 concentration was evaluated after 100 min.

ASSOCIATED CONTENT Supporting Information. Sequences of the oligonucleotides used in this study are available in Table S1.

ABBREVIATIONS CRISPR/Cas, Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated systems (Cas) ; PFGE, pulse field gel electrophoresis ; TREC, tandem repeat coupled with endonuclease cleavage ; TREC-IN, TREC-assisted gene knock-in ; PAM, protospacer adjacent motif.

AUTHOR INFORMATION Corresponding author *Tel: (33) 5 57 12 23 59; Fax: (33) 5 57 12 23 69; E-mail: [email protected]. Author contribution I.T., C.L. and P.S.P. designed the experiments; I.T. performed the experiments with the help of C.L., G.G.; P.S.P. and I.T. wrote the manuscript with the improvements from C.L., A.B., J.J. and S.V. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors thank Luis Serrano and Maria Lluch-Senar for useful discussions. This work was supported in part by the US National Science Foundation [grant number IOS-1110151] and by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 634942. We also thank Yanina Veronica Valverde Timana and Marie-Pierre Dubranat for technical help.

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(19) Kouprina, N., and Larionov, V. (2008) Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat. Protoc. 3, 371–377. FIGURE LEGENDS

Figure 1. Construction of the gRNA expression vector and glpO deletion design. a. Design of gRNA expression constructs. Expression of chimeric gRNA is controlled by the snoRNA SNR52 promoter and terminator from the 3’ region of the yeast SUP4 gene. The CAN1.Y target 20 nt sequence from the original plasmid from DiCarlo et al. was replaced to generate the other plasmids b. Schematic for the seamless cloning of the glpO guide sequence oligonucleotides into the customized p426-SNR52p-AarISUP4t plasmid. The type IIS AarI restriction enzyme recognition and cleavage sites are indicated in orange and by arrowheads, respectively. The glpO guide oligonucleotides are annealed and contain overhangs for ligation into the pair of AarI sites in pgRNA.AarI. c. Localization of the 20 nt-guide sequence within the glpO gene. Adjacent PAM sequence tgg is highlighted in yellow. Sequence of the 90 bp-recombination template for the deletion of the glpO gene in Mmc is shown in gray. Figure 2. Screening yeast for glpO-deleted mycoplasma genomes. a. Genomic DNA from pools of 20 yeast colonies co-transformed with the pgRNA.glpO plasmid and recombination template was extracted for PCR screening for the glpO deletion. b. Schematic of the glpO region in Mmc (wt) and glpO-deleted mutants (ΔglpO). Lengths of PCR products are indicated. c. Representative results of the PCR screen. Pools with bands of about 500 bp indicated the presence of ΔglpO mutants. d. Gel electrophoresis of PCR products obtained from individual clones present in the positive pools 7 and 8. e. Gel electrophoresis of multiplex PCR to check mycoplasma genome integrity of mutants P7.10, P7.14, P8.18 and P8.20. M, 100 bp-ladder (Promega); wt, positive control DNA from Mmc; (-), H2O negative control. F. PFGE analysis of mutants P7.10, P7.14, P8.18 and P8.20 after PspXI digestion; M, CHEF S. cerevisiae chromosomal DNA (Biorad); wt, positive control from wt Mmc.

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Figure 1. Construction of the gRNA expression vector and glpO deletion design. a. Design of gRNA expression constructs. Expression of chimeric gRNA is controlled by the snoRNA SNR52 promoter and terminator from the 3’ region of the yeast SUP4 gene. The CAN1.Y target 20 nt sequence from the original plasmid from DiCarlo et al. was replaced to generate the other plasmids b. Schematic for the seamless cloning of the glpO guide sequence oligonucleotides into the customized p426-SNR52p-AarI-SUP4t plasmid. The type IIS AarI restriction enzyme recognition and cleavage sites are indicated in orange and by arrowheads, respectively. The glpO guide oligonucleotides are annealed and contain overhangs for ligation into the pair of AarI sites in pgRNA.AarI. c. Localization of the 20 ntguide sequence within the glpO gene. Adjacent PAM sequence tgg is highlighted in yellow. Sequence of the 90 bp-recombination template for the deletion of the glpO gene in Mmc is shown in gray.


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Figure 2. Screening yeast for glpO-deleted mycoplasma genomes. a. Genomic DNA from pools of 20 yeast colonies co-transformed with the pgRNA.glpO plasmid and recombination template was extracted for PCR screening for the glpO deletion. b. Schematic of the glpO region in Mmc (wt) and glpO-deleted mutants (ΔglpO). Lengths of PCR products are indicated. c. Representative results of the PCR screen. Pools with bands of about 500 bp indicated the presence of ΔglpO mutants. d. Gel electrophoresis of PCR products obtained from individual clones present in the positive pools 7 and 8. e. Gel electrophoresis of multiplex PCR to check mycoplasma genome integrity of mutants P7.10, P7.14, P8.18 and P8.20. M, 100 bp-ladder (Promega); wt, positive control DNA from Mmc; (-), H2O negative control. F. PFGE analysis of mutants P7.10, P7.14, P8.18 and P8.20 after PspXI digestion; M, CHEF S. cerevisiae chromosomal DNA (Biorad); wt, positive control from wt Mmc.


Cas9

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