Cas9 Genome Editing and Gene Regulation

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Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation using Csy4 in Saccharomyces cerevisiae Raphael Ferreira, Christos Skrekas, Jens Nielsen, and Florian David ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00259 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation using Csy4 in Saccharomyces cerevisiae Raphael Ferreira1, 2, Christos Skrekas1, Jens Nielsen1, 2, 3 and Florian David1, 2, * 1 Department of Biology and Biological Engineering, Chalmers University of Technology, SE412 96 Gothenburg, Sweden 2 Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE412 96 Gothenburg, Sweden 3 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK2800 Kgs. Lyngby, Denmark * Corresponding author

Abstract The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made towards developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome editing and gene interference applications in S. cerevisiae. In regards to genome editing, we performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out of 24 colonies tested. Then, we used this system to efficiently transcriptionally regulate the 3 genes, OLE1, HMG1 and ACS1. Thus, we demonstrate that multiplexed genome editing and gene regulation can be performed in a fast and effective manner using Csy4. Keywords: CRISPR; Csy4; Multiplexing; Metabolic Engineering; Gene editing; CRISPRi; Gene Regulation;

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INTRODUCTION The advent of molecular biology has brought a myriad of ingenious tools to manipulate and build complex genetic elements. In particular, the recent type II CRISPR technology, adapted from Streptococcus pyogenes, has proven to be highly effective for editing and regulating genes in various organisms (Mali et al. 2013, DiCarlo et al. 2013, Zalatan et al. 2015). The technology relies on the combination of an endonuclease, CRISPR associated protein 9 (Cas9), and a guide-RNA (gRNA), which jointly form the complex of Cas9:gRNA (Jinek et al. 2012). Cas9 is guided by the gRNA to a 20 nucleotides (nt) sequence followed by a 3 nt “NGG” called Protospacer Adjacent Motif (PAM) (Bolotin et al. 2005). When bound to the target, Cas9 cleaves the DNA sequence 3nt upstream the PAM, activating the endogenous repair mechanism, such as homologous recombination (HR) or non-homologous end joining (NHEJ), when the complex is oriented towards genomic DNA (Marraffini et al. 2010). Altogether, the system has been shown to generate knockouts and knockins at an unprecedented pace and specificity in a multitude of organisms (Sander et al. 2014). In S. cerevisiae, Mans et al. have performed up to six gene deletions in a single experiment (Mans et al. 2015). A catalytically inactive version of Cas9 (dCas9) has shown regulatory control of gene expression by obstructing the transcriptional machinery (Gilbert et al. 2013). Additionally, dCas9 repression can be further enhanced by fusing it with repressive domains, such as the mammalian transcriptional repressor domain Mxi1 (Gilbert et al. 2014). As such, Gander et al. have recently exploited dCas9-Mxi1 repressive mechanism to effectively built up to seven layers of synthetic NOR gates circuits, in S. cerevisiae (Gander et al. 2017). Likewise, dCas9 can be coupled to activator transcription factor domains, such as the tripartite VP64-p65-Rta (VPR) which has been extensively characterized as an efficient tool for positively interfering gene regulation (Chavez et al. 2015, Smith et al. 2016, Jensen et al. 2017). Expressing multiple gRNAs in a single construct generally requires using the same promoter several times, ultimately causing various issues in terms of cloning as well as reaching robust transcriptional expression of the gRNAs. Consequently, Nissim et al. have exploited the type III CRISPR/Cas-associated Csy4 endoribonuclease from Pseudomonas aeruginosa, in mammalian cells. They achieved multiple gene regulations by cleaving mRNAs consisting of gRNA sequences flanked by recognition motifs for Csy4 (Nissim et al. 2014). Also, the bacterial origin of Csy4 makes it an ideal orthogonal tool to build complex synthetic circuits without interfering with the endogenous RNA machinery of the host cell (Nissim et al. 2014, Ferry et al. 2017). While the functionality of Csy4 has been shown to operate in several host cells (Qi et al., 2012), it has so far not been characterized for genome editing and gene regulation in S. cerevisiae. Here, we propose to exploit the Csy4 RNA processing capacity to generate multiple gRNAs from a single transcript for genome editing and gene regulation applications in S. cerevisiae. In regards to genome editing, we performed a FAA1, FAA4, POX1 and TES1 quadruple knockouts with 96% efficiency out of 24 colonies tested. Then, we characterized Csy4 for CRISPRi applications by efficiently regulating gene expression of 3 genes, namely OLE1, HMG1 and ACS1. Our study presents a robust, orthogonal and inducible CRISPR platform that will be valuable for accelerated strain engineering applications, as well as, fine-tuning of biosynthetic pathways in yeast.


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RESULTS The Csy4 endoribonuclease from Pseudomonas aeruginosa has a high degree of substrate specificity towards a 28 nucleotides RNA stem-loop (5’GTTCACTGCCGTATAGGCAGCTAAGAAA-3’), which we termed “28nt” (Haurwitz et al. 2010, Haurwitz et al. 2012). Once bound to the RNA stem-loop, Csy4 cleaves after the guanine at position 20 (G20), allowing to generate multiple RNA transcripts. We first sought to characterize the ability to perform multiple gene knockouts by simultaneously generating gRNAs from a single transcript using Csy4 in yeast. Thus, starting from the IMX581 strain (MATa MAL2‐8c SUC2 ura3‐52 can1::PTEF1-cas9), we genomically integrated Csy4, with a N-terminal SV40 NLS peptide signal, under the expression of the constitutive active TEF1 promoter (Fig. 2A). We designed an array of four gRNAs in a row, targeting genes involved in the fatty acid metabolism: FAA1, FAA4, POX1 and TES1. The gRNAs transcript was expressed under the constitutive RNA Pol. III promoter, SNR52, in a multi-copy 2µ plasmid. The plasmid was co-transformed with linear repair DNA fragments matching the flanking of the genes targeted, resulting to a complete loop-out of the CDS regions. Notably, when tested with two gRNAs in a row, co-expressing the gRNAs with Csy4 allowed 100% knockout efficiency, while its absence (negative control) still allowed single and double deletion up to 38% and 50% respectively for 8 colonies tested each (Fig. 2B). When the expression was extended to 4 gRNAs in a row, the presence of Csy4 reached close to 100% (96%) efficiency with only one colony out of 24 getting only 3 out of 4 genes deleted (Table S6). The absence of Csy4 did not lead to quadruple nor triple deletions but showed double deletions with 100% efficiency (Fig. 2C, Table S6). Based on these experiments, Cas9 seems to be able to correctly cut the DNA targets mediated by the first two gRNAs, even if they are expressed as one transcript in combination with other gRNAs and independent from subsequent Csy4 processing (Fig. 2B, 2C). Here, we speculate that the gRNAs being expressed with a multicopy plasmid (~50 plasmids/cell), under a constitutive promoter, along with presence of repetitive elements (28nt and the 79nt gRNA scaffold) in the array have most likely increase the probability of incomplete or partially degraded transcripts. More importantly, several studies have pointed out Cas9 flexibility in the ability to process DNA with long gRNA (Zalatan et al. 2015, Bao et al. 2014). Altogether, these details could explain gene knockouts mediated by the first two gRNAs even in the absence of Csy4. Briefly, Csy4-mediated multiplexed genome editing strategy shows equivalent, or improved, efficiency than previous studies (Fig. S4), as well as, separate expression of each gRNA (Table S5). Next, we assessed the capacity of the platform for CRISPRi applications. We used dCas9 fused to the tripartite activator VPR at the C-terminus. We cloned a series of 3 gRNAs targeting OLE1, HMG1 and ACS1 promoter regions which we individually characterized in a previous study (Jensen et al. 2017). Those genes are involved in crucial pathways and incorrect regulation is known to severely affect the growth of the cell, thus stressing the importance of fine-tuning the expression. Similar to the knockout experiment, the gRNAs were separated with the 28 nt motif, and expressed under a tetracycline inducible RPR1 promoter in a single copy plasmid (Smith et al. 2016). The inducible platform allows better control of the gRNA expression, which offers a major advantage when targeting essential



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genes, such as OLE1, as potential growth defects might occur by having the gRNA constitutively expressed. We expressed Csy4 under the TEF1 promoter, using a single copy plasmid. In order to characterize the effect of the gRNAs, we constructed three strains carrying either HMG1, OLE1 or ACS1 promoter coupled to GFP. In order to assay the potential bias of the positioning within the transcript, we constructed a series of 3 plasmids where each gRNA was located at different positions (Fig. 3A). For OLE1 and HMG1, the multiplexed and Csy4 processed gRNAs reached the same level of transcriptional regulation as when the particular gRNA was expressed alone. For ACS1, the multiplexed gRNAs reached almost the same level as the positive control in presence of Csy4 (Fig. 3B). In contrast to the background activity in the gene deletion experiment, expressing the multiplexed gRNA transcript in absence of Csy4 did not affect the GFP expression (Fig. 3B), which seems to highlight the importance of expressing gRNAs under a controlled abundance in order to achieve significant interference. Furthermore, we were able to prove robust and stable gene perturbation based on stable expression and Csy4-mediated processing of gRNAs cassettes (Fig. S1-S3).

CONCLUSION In conclusion, the RNA processing capacities of Csy4 can be efficiently applied in S. cerevisiae for gene deletion and interference. We developed a series of strains and plasmids that can be readily used for the above-mentioned purposes. The CRISPRi inducible tool permits the control of the expression of multiple genes at the same time, which we envision will become valuable tool for combinatorial expression of gRNAs for multiplexed fine-tuning of target gene expression.


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Methods and Methods Plasmid and strain construction Plasmids, strains, gRNA sequences and primers used in this study are listed in Tables S1-S4. The plasmid maps, generated with Benchling, are provided. Oligonucleotides were ordered from Eurofins and IDT. Csy4NLS (SV40 NLS sequence: 5’-gccccaaagaagaagagaaaagttaga3’) was ordered as a synthetic codon optimized gene from Genscript (Piscataway, NJ). S. cerevisiae transformations were done using lithium acetate and PEG3350 (Gietz and Woods 2006). All constructs were cloned using Gibson assembly (Gibson et al. 2009). Extraction of genomic DNA for colony PCR was followed as previously described (Lõoke et al. 2011). For the multiple gene knockouts, IMX581 and the plasmid pMEL13 were obtained from EUROSCARF (Frankfurt, Germany). Csy4 coding sequence was amplified and inserted into the integrative chromosome X pCfB393 plasmid vector (Jensen et al. 2014) using Csy4NLSp393-F/R and p393-F/R set of primers, resulting to p393_Csy4NLS. The plasmid was digested with FastDigest-NotI, purified and transformed into IMX581. Correct integration into the chromosome X was screened by colony PCR using primers X2check-F, X2check-R and X2csy4-R. The URA3 marker was removed using the Cre-expressing plasmid pSH65, as previously described (Güldener et al. 1996), yielding the new strain Csy4_Cas9. The double gRNAs plasmid (pCRA01) was constructed using pMEL-F/R, FAA1-F/R and FAA4-F/R set of primers. From this vector, the remaining two gRNAs were added to form the quadruple gRNAs plasmid (pCR02 with the pMEL-FAA-F and pMEL-FAA-R, TES-F/TESR and POX-F/POX-R set of primers. The diagnostic primers, the repair oligos and the targetgRNA for the individual characterization were designed using the Yeastriction webtool (http://yeastriction.tnw.tudelft.nl). For the single gRNA individual characterization, gRNAs were cloned into the pROS13 or recombined in vivo with linearized pMEL13 vector (Mans et al. 2015). Co-transformation of the plasmid expressing the gRNAs and the different repair fragments resulted in the in vivo double strand cut in targeted genes by the Cas9 nuclease, which ultimately allowed the integration of the repair fragments by HDR (Ferreira et al. 2017). The knockouts were screened for complete loop-out by using the correspondent diagnostic primers designed to bind outside the CDS and confirmed on gel agarose. For CRISPRi experiments, Csy4NLS was amplified using 413Csy4-F/R primers and cloned into the XbaI & XhoI digested p413TEF vector backbone via Gibson assembly, leading to p413_Csy4NLS plasmid. The inducible plasmid dCas9-VPR plasmid pDTU-113 (Jensen et al. 2017) was modified using dCas9v2-F/R, resulting to dCas9v2. Three cassettes, containing the different gRNAs separated with the 28nt located at different positions, were ordered from Genscript (Piscataway, NJ, USA). Then the cassettes were amplified with the primers gRNAF and gRNA-R and cloned into dCas9v2 vector leading to plasmids pCRA03, pCRA04 and pCRA05. ACS1 single gRNA expression plasmid was constructed using ACS1-gRNA-F/R oligos inserted into pDTU-113. Single gRNA plasmids for HMG1 and OLE1 were constructed prior this study (Jensen et al. 2017) using similar strategy as ACS1. For the different GFP strains, ACS1, OLE1 and HMG1 promoter regions, i.e. 1000nt upstream of the start codon, were amplified from CEN.PK113-11C genomic DNA using ACS1p-395-F/R,



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OLE1p-395-F/R and HMG1-395-F/4 set of primers, respectively. The products were cloned into pCfB395(GFP) leading to PACS1-GFP, POLE1-GFP and PHMG1-GFP. The different vectors were digested with FastDigest-NotI (ThermoFisher), purified and transformed into the CEN.PK113-11C (Kötter, University of Frankfurt, Germany). Then, the HIS3 marker was removed using the Cre-expressing plasmid pSH47, as previously described (Güldener et al. 1996), yielding the new strains CRA01, CRA02 and CRA03. These strains were then cotransformed with pCRA03, pCRA04 and pCR05 together with p413_CSY4NLS or p413TEF for the negative control, leading to the new strains CRA06 to CRA29.

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Growth medium S. cerevisiae strains with uracil and histidine auxotrophies were grown on YPD plates containing 20 g·L-1 glucose, 10 g·L-1 yeast extract, 20 g·L-1 peptone from casein and 20 g·L-1 agar. URA3 and HIS3 plasmid carrying strains were grown on selective growth medium containing 6.9 g·L-1 yeast nitrogen base w/o amino acids (Formedium, Hunstanton, UK), 0.77 g·L-1 complete supplement mixture w/o histidine and uracil (Formedium), 20 g·L-1 glucose and 20 g·L-1 agar. KanMX plasmid carrying strains were selected with 10 g·L−1 Bacto yeast extract, 20 g·L-1 Bacto peptone, 20g·L-1 agar), supplemented with 200 mg·L-1 G418 (Geneticin®). Pre-cultures for fluorescence measurement were cultured in minimal medium containing 20 g·L-1 glucose, 5 g·L-1, (NH4)2SO4, 14.4 g·L-1 KH2PO4, 0.5 g·L-1 MgSO4·7H2O. After sterilization, 2 mL·L-1 trace element solution and 1 mL·L-1 of vitamin solution were added (Verduyn et al., 1992).

Fluorescence measurements For real-time monitoring of GFP expression, BioLector® was used (m2p-labs GmbH, Baesweiler, Germany). The cultures were grown in using FlowerPlates®, the total volume of each culture was 1 mL minimal media and initial OD600 was 0,05. The inducible system was activated by adding 250 ng·mL-1 of aTc into the 1 mL culture. GFP expression levels were measured by green fluorescence units in function of OD600 units.


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Figure 1. Graphical abstract of the study. Csy4 (purple) recognizes a 28 ribonucleotides stem-loop sequence and cleave at position 20 (red arrow). This feature allows to generate multiple RNA segments which we presently use for transcribed multiple gRNAs in a single transcript. The gRNA (blue and red) can then be processed for different purposes.



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Figure 2. Csy4 characterization for multiplexed gene knockouts. A. Graphical representation of the experiment outline. The experiments were carried out in a strain overexpressing genes encoding for cas9 and csy4. The different gRNAs were expressed under the constitutive RNA Pol.III, SNR52, using a multi-copy plasmid. The gRNAs were targeting FAA1, FAA4, TES1 and POX1 with gRNA in the first position targeting POX1, gRNA in the second position targeting TES1, gRNA in the third position targeting FAA1, and gRNA in the fourth position targeting FAA4. The plasmids were transformed along with repair DNA fragment matching the flanking regions of each gene, which ultimately resulted in a complete loop-out of the coding sequence via the endogenous homologous recombination repair mechanism B. Results from the double deletion experiment. As a negative control, IMX581 strain without Csy4, was used. Expressing the gRNAs jointly with Csy4 resulted in 100% double deletion efficiency with 8 colonies tested. Out of 8 colonies tested for the negative control, 4 showed double deletion, 3 showed single deletion, and 1 colony showed zero deletion. C. In the 4 gRNAs experiment, the expression of the gRNAs together with Csy4 reached 96% knockout efficiency for quadruple deletion (24 colonies tested). In absence of Csy4, no colonies with quadruple deletion were found but from the 8 colonies tested 100% had 2 genes deleted.


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Figure 3. Characterization for multiplexed gene regulation with CRISPRi. A. Strains expressing GFP under OLE1, HMG1 or ACS1 promoters were co-transformed with the different multiplexed plasmids together with a plasmid expressing csy4, or an empty plasmid for the negative control. pCRA05 expresses gRNAs in the following order: OLE1 gRNA, HMG1 gRNA, and ACS1 gRNA. pCRA06 expresses gRNAs in the following order: ACS1 gRNA, OLE1 gRNA, and HMG1 gRNA. pCRA07 expresses gRNAs in the following order: HMG1 gRNA, ACS1 gRNA, and OLE1 gRNA. B. Fluorescence values retrieved from the different transformant strains at an OD600 of 8. For POLE1-GFP experiment, the multiplexed plasmids showed a similar expression as the gRNA expressed alone, only when csy4 was expressed. The multiplexed plasmids without csy4 expressed showed similar expression level of GFP as the control (empty plasmid) (Fig. S1). C. For PACS1-GFP experiment, the multiplexed plasmid showed almost the same expression as the gRNA expressed alone when csy4 was expressed. The multiplexed plasmids without csy4 expressed showed similar expression level of GFP as the control (empty plasmid). D. For PHMG1-GFP experiment, the multiplexed plasmids showed a similar expression as the gRNA expressed alone, only when csy4 was expressed. The multiplexed plasmids without csy4 expressed showed similar expression level of GFP as the control (empty plasmid). All experiments were done with quadruplicate biological replicates monitored with a Biolector over 60h. The empty plasmid



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strain expresses dCas9-VPR plasmid together with an empty p413TEF plasmid. Multiplexed gRNAs (01, 02 and 03) corresponding to pCRA05, pCRA06 and pCRA07 were expressed with or without the p413_Csy4NLS plasmid.


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Supporting Information Figure S1-3: CRISPRi transcriptional regulation using Csy4; Figure S4: Plasmid stability assessment, Figure S5: Genome editing example scheme and individual gRNA characterization. Table S1, list of plasmids used in this study; Table S2, list of strains used in this study; Table S3, list of gRNA sequences used in this study; Table S4, list of oligos used in this study; Table S5: Method comparison with Bao et al. 2014; Table S6: Summary of gene deletion experiment.



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Author Contributions RF, JN and FD conceived this project. RF and FD designed all the experiments. RF and CS performed the experiments. All authors analyzed the data. RF wrote the paper. All authors read and approved the final manuscript. Notes The authors declare no competing financial interest. Acknowledgements The authors would like to acknowledge the Novo Nordisk Foundation, the Knut and Alice Wallenberg Foundation, and the Swedish Foundation for Strategic Research for funding support.


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A

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1 PSNR52 (Pol. III) 2 cas9 csy4 3 FAA1 FAA4 POX1 TES1 FAA1 FAA4 4 genome integrated !"#$%*%!#)+ gRNA gRNA gRNA gRNA gRNA gRNA 5 Csy4 Cas9 6 7 URA3 URA3 8 !"#$%$" 9 &$'$($& 10 !"#$%*%!#)+ 11 )"#$%$" 12 !"#$%$" &$'$($& 13 &$'$($& %# $ 14 )"#$%$" *"#$%$+" 15 &$'$($& &$'$($& !"#$%*%!#)+ !"#$%*%!#)+ 16 *"#$%$+" !(( # $ 17 &$'$($& %# $ 18 genome !"#$%$" FAA1 FAA4 POX1 TES1 ,"#$%$+" &$'$($& 19 &$'$($& Chr. XV Chr. XIII Chr. VII 20 )"#$%$" Chr. X &' # $ -"#$%$+" &$'$($& 21 &$'$($& *"#$%$+" 22 &$'$($& 23 !(( # $ !"#$%&'() &' # $ 24 2 gRNAs experiment 4 gRNAs experiment !"#$%&'() 25 26 Cas9 +!"#$%*%!#)+ Csy4 Cas9 only 0 gene !"#$%&'() !"#$%&'() !"#$%$" Cas9 +!"#$%*%!#)+ Csy4 Cas9 only 27 0 gene deleted !"#$%$" &$'$($& !"#$%$" 28 deleted &$'$($& !" # $ %# $ 4% &$'$($& 1 gene 29 )"#$%$" !"#$%$" )"#$%$" !"#$%$" deleted &$'$($& &$'$($& !" # $ 12% &$'$($& &$'$($& 30 1 gene 2 genes *"#$%$+" )"#$%$" 31 )"#$%$" )"#$%$" &$'$($& &$'$($& deleted '( # $ '( # $ deleted 50% &$'$($& 32 n=7 &$'$($& &$'$($& ,"#$%$+" %&# $ *"#$%$+" 3 genes 38% 33 &$'$($& %&# $ &$'$($& *"#$%$+" !"" # $ !(( # $ deleted 100% 100% &' # $ 34 -"#$%$+" 96% &$'$($& ACS Paragon Plus Environment !"" # $ 2 genes &$'$($& &$'$($& *"#$%$+" 4 genes 35 deleted n=8 n=8 n=24 n=8 deleted 36 &$'$($&

PSNR52 (Pol. III)

B

A

BB


OR

1200 1000 800 600 400 200 0

OR

+

C

OLE1 pCR05 pCR06 pCR07 (empty w/o Csy4 plasmid)

OLE1 gRNA alone

pCR05

pCR06

with Csy4

pCR07

GFP expression for PACS1-GFP

1400 1200

[GFP]

1000 800 600 400 200 0

ACS1 pCR05 pCR06 pCR07 (empty w/o Csy4 plasmid)

D

ACS1 gRNA alone

pCR05

pCR06

with Csy4

pCR07

GFP expression for PHMG1-GFP

600 500 400

[GFP]

+

Page 18 of 18

1400

[GFP]

1 PRPR1 (Inducible Pol. III) 2 3 OLE1 HMG1 ACS1 4pCRA05 gRNA gRNA gRNA 5 6 7 8pCRA06 ACS1 OLE1 HMG1 9 gRNA gRNA gRNA 10 11 12 pCRA07 HMG1 ACS1 OLE1 13 gRNA gRNA gRNA 14 15 16 URA3 cas9 dcas9-VPR 17 18 19 20 PTEF1 21 22 23 csy4 24 25 26 HIS3 27 28 29 30 31 32 PHMG1, PACS1 or POLE1 33 34 GFP 35 genome integrated 36 37 38 39 40 41 42 43 44

GFP expression for POLE1-GFP

300 200 100

0 ACS Paragon PlusHMG1 Environment pCR05 (empty plasmid)

pCR06

w/o Csy4

pCR07

HMG1 gRNA alone

pCR05

pCR06

with Csy4

pCR07

Cas9 Genome Editing and Gene Regulation

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