Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast

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Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast Nicholas S McCarty, William M Shaw, Tom Ellis, and Rodrigo Ledesma-Amaro ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00041 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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ACS Synthetic Biology

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Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast

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Nicholas S. McCarty1,2, William M. Shaw2,3, Tom Ellis2,3 & Rodrigo Ledesma-Amaro2,3,4,*

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1 Bioengineering, California Institute of Technology, Pasadena, CA 91125, USA

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*Correspondence: [email protected]

2 Imperial College Centre for Synthetic Biology, Imperial College London, London, SW7 2AZ, UK 3 Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK 4 Lead contact

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ABSTRACT

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CRISPR is a versatile technology for genomic editing and regulation, but the

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expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present

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here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly

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of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction

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enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and

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expressed from a single transcriptional unit. We demonstrate that these gRNA arrays

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can stably regulate gene expression in a synergistic manner via dCas9-mediated

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repression. This approach expands the number of gRNAs that can be expressed in

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this model organism and may enable the versatile editing or transcriptional regulation

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of a greater number of genes in vivo.

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Keywords: yeast, MoClo toolkit, CRISPR, multiplex, assembly method, Golden Gate

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assembly

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The expression of many gRNAs simultaneously enables multiplexed genome

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engineering and transcriptional modulation of genetic targets with CRISPR-Cas

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systems. There are numerous methods to accomplish this, such as by encoding each

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gRNA under the control of a separate RNA polymerase III (Pol III) promoter1, cleaving

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gRNAs from a longer RNA sequence by encoding them in introns2, by flanking gRNAs

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with self-processing ribozyme sequences3, or via excision of gRNAs by other Cas

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family endonucleases4–6. This last approach, in which gRNAs are excised by an

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endonuclease, can be performed with Csy4, which processes pre-crRNA in native

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CRISPR systems7. By flanking gRNAs with Csy4 recognition sites, multiple crRNAs or

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fused gRNAs can be efficiently processed from a single transcript in a variety of cell

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lines5,6.

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Numerous methods have also been reported to assemble gRNA arrays for in vitro and

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in vivo applications, typically by utilizing annealed oligonucleotides with modified

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Gibson or Golden Gate assembly approaches6,8–12. These studies have largely

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focused on mammalian cells but methods have recently emerged for gRNA

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multiplexing in yeast. Efforts to multiplex gRNAs in S. cerevisiae have been limited to

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7 gRNAs if expressed from individual promoters13 and 4 gRNAs if expressed and

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processed from a single array, either by using gRNA-tRNA arrays with Pol III

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promoters or by using Pol II promoters with the Csy4 enzyme to process the gRNA

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arrays.5,14

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Here, we expand the widely-used Yeast MoClo Toolkit1, a widely-used library of highly

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characterized parts designed for Golden Gate assembly and integration into S.

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cerevisiae, to enable the rapid, modular assembly and expression of gRNA arrays in

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this model organism. This method is a dual-PCR, Type IIs restriction enzyme Golden

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Gate approach that enables the construction of up to 12 gRNAs in a single array, each

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flanked by recognition sites for Csy4, in two days. This method uses one plasmid to

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produce gRNAs with desired spacer sequences and a second plasmid to assemble

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the final arrays (Supplementary Figure S1). All other parts are available from the

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Yeast MoClo Toolkit.1 This system will enhance the ease and utility of multiplexed

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gRNA applications in S. cerevisiae.

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RESULTS

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We build and express arrays of tandem gRNAs in S. cerevisiae by adding just two

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parts to the Yeast MoClo Toolkit: a Guide-Generating Vector (GGV) and a Destination

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Vector (DV) (Figure 1a and Supplementary Figures S2, S3). An initial round of PCR

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using the GGV as a template adds desired spacer sequences via primer overhangs.

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The GGV contains a Csy4 recognition site, a selection/screening marker and a

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sequence encoding the gRNA scaffold of interest (which associates the gRNA with a

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chosen Cas endonuclease). Once the linear PCR products are ligated, the designed

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spacers are positioned next to the scaffold to produce functional gRNAs.


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A second round of PCR adds Type IIs restriction enzyme recognition sites with unique,

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four-nucleotide overhangs via primer overhangs15. The generated, linear PCR

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fragments can then be assembled into the DV via Golden Gate assembly, which

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consists of a promoter of interest, followed by another screening/selection marker, a

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terminal copy of the Csy4 recognition sequence, and the terminator of interest (Figure

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1a). Target sequences of gRNAs in the array must be devoid of BsmBI, BsaI and NotI

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recognition sites. For this study, we use a DV with a start codon-deficient variant of

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PTDH3, a Pol II promoter derived from the Yeast MoClo Toolkit1. The DV also contains

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designed XhoI and BglII sites after the promoter and before the final Csy4 site,

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respectively, which enables assembled gRNA arrays to be swapped between

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promoters and terminators from the Yeast MoClo Toolkit.

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Figure 1: Assembly of gRNA arrays and screening of gRNA candidates. (a) Schematic overview of the system for assembling gRNA arrays. Primers are indicated by arrows, with slanted lines indicating primer overhangs. (b) Spatial positions of the spacer sequences tested on their corresponding genomic targets. Each gRNA contains a 20-nucleotide spacer sequence complementary to the PALD6, PHHF1 or PTEF1 promoter and is adjacent to an NGG PAM sequence. Numbers in the gray boxes indicate each unique gRNA tested and corresponds to the results plotted in panel (c). Numbers marked with an asterisk indicate gRNAs which were used in the construction of gRNA arrays. A minimal number of PAM sites



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in the PTEF1 promoter limited the number of gRNAs which could be tested for mTagBFP2. (c) Normalized fluorescence for each individual gRNA tested. Each sample was normalized to the fluorescence measured for a yeast strain expressing a null spacer sequence (100% offtarget score using Doench et al. algorithm)16. The fluorescence of yeast expressing the null gRNA was set to a value of 1.0 for each fluorescent reporter. All other groups were normalized to the null gRNA mean. Error bars represent the standard deviation between the n=3 replicates within each group.

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To assess the efficiency of gRNA array assembly using this system, arrays containing

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between 3 to 12 gRNAs within a single transcriptional unit were constructed. Colonies

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were screened for the absence of the screening marker (GFP) (Supplementary

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Figure S4) and the insert length of each construct was evaluated by diagnostic

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restriction digest with BsaI (Supplementary Figure S5). Restriction digests indicated

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that 100% of colonies contained an array of the expected size (for 3 or 6 gRNAs),

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followed by a decrease in efficiency for 9 and 12 gRNA arrays (66.6% and 50%,

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respectively) (Supplementary Figure S6). Purified DNA isolated from colonies that

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yielded bands of the expected sizes from restriction digests were additionally sent

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for Sanger sequencing (n=3 each) for the 3, 6, 9 and 12 gRNA arrays. All samples

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were sequence-verified without any observed mutations (Supplementary Methods).

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To demonstrate gRNA multiplexing capacity, an experiment was implemented that

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enabled us to test the transcriptional repression mediated by multiple gRNAs in

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budding yeast simultaneously using fluorescent reporters and flow cytometry. S.

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cerevisiae strain BY4741 was engineered to express three fluorescent reporters, each

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driven by a unique constitutive promoter: PTEF1-mTagBFP2, PHHF1-mRuby2, and PALD6-

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Venus. These promoters are constitutive, strong promoters from the Yeast MoClo

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Toolkit derived from genes in S. cerevisiae that are either nonessential or have

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paralogs in the genome. These promoter-reporter pairs were cloned into a single

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cassette and genome-integrated at the HO locus. A DNA construct expressing dCas9

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(with N- and C-termini nuclear localization signals) and Csy4 endonuclease, under

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control of PPGK1 and PHHF2, respectively, were also genome-integrated at the LEU2

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locus17.

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Individual gRNAs were next designed to repress fluorescent protein expression by

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targeting each of the three constitutive promoters and their individual efficiencies were

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assessed. Spacer sequences to repress PTEF1, PHHF1, or PALD6 were targeted to bind


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between positions -300 and +1 bp upstream of the start codon of each fluorescent

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reporter (Figure 1b). The BY4741-derived strain expressing these fluorescent

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reporters, dCas9 and Csy4 was then transformed with individual gRNAs possessing

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the designed spacer sequences integrated at the URA3 locus. Changes in

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fluorescence of each reporter following integration of the gRNA were assessed by flow

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cytometry (Figure 1c).

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Using these data, four gRNAs were selected for dCas9-mediated transcriptional

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repression for each of the fluorescent reporters. They were selected based on weak

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repression of fluorescent output, so that synergistic effects could be visualized when

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multiplexing many gRNAs to a promoter, and distributed targeting within the promoter

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regions so that gRNA recognition sites do not overlap, as we speculated that this would

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occlude our ability to observe synergistic dCas9-mediated repression of multiple

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targets simultaneously. gRNAs V1, V4, V6, V8 for Venus repression, gRNAs R2, R8,

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R6, R4 for mRuby2 repression, and gRNAs B1, B2, B3, B4 for mTagBFP2 repression

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(Figure 1b) were selected.

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Arrays encoding 3, 6, 9, or 12 gRNAs within a single transcriptional unit were

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constructed. As arrays increase in size, more gRNAs are targeted to repress

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fluorescent protein expression; the 3-gRNA array targets one gRNA per promoter,

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whereas the 12-gRNA array targets four gRNAs to each promoter. The 3-gRNA array

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consists of V1, R2 and B1. The 6-gRNA array consists of V1, R2, B1, V4, R8 and B2.

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The 9-gRNA array consists of V1, R2, B1, V4, R8, B2, V6, R6 and B3. The 12-gRNA

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array consists of all twelve selected gRNAs. All arrays were genome-integrated at the

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URA3 locus (Figure 2a). We observed a slight growth defect in the strains expressing

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gRNA arrays compared to controls, potentially due to a side targeting of the three

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native promoters.18

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Characterization of per cell fluorescence by flow cytometry revealed synergistic

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repression of all three fluorescent proteins when an increasing number of gRNAs were

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expressed from the arrays (Figure 2b). Co-expression of dCas9 with four gRNAs

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targeting each fluorescent reporter, in the case of the 12-gRNA array, showed a

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repression of 92% in the mVenus channel, 81% in the mRuby2 channel and 95% in

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the mTagBFP2 channel. These repression efficiencies are significantly greater than



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with any of the individual gRNAs and indicated synergistic repression of three separate

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genes by a tandem array of up to 12 gRNAs using this multiplexed system.

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Since homologous recombination in budding yeast is more active in regions containing

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repetitive DNA sequences19, we also assessed the stability of these repetitive gRNA

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arrays over generations of growth by performing flow cytometry on yeast expressing

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each gRNA array after five consecutive, 1:100 dilutions with twelve hours of growth

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between dilutions; 72 hours of growth in total (Supplementary Figure S7). Flow

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cytometry

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transcriptional repression of fluorescent protein expression. Colony PCR on the gRNA

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arrays at the URA3 locus confirmed stable genomic integration of all gRNA arrays after

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the dilution series (Supplementary Figure S7).

after

five

consecutive

dilutions

indicated

sustained,

synergistic

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Figure 2: Genome-integrated gRNA arrays in S. cerevisiae enable synergistic, dCas9mediated repression of gene expression. (a) General method to quantify the repression efficiency of tandem gRNA arrays in S. cerevisiae. gRNA arrays were driven by PTDH3 on a plasmid integrated at the URA3 locus. Arrays contain 3, 6, 9, or 12 gRNAs in tandem, with each successive increase in tandem length adding an additional gRNA targeting each of the promoters driving fluorescent protein expression. gRNA arrays were transformed into yeast expressing Csy4, dCas9 and Venus, mRuby2 and mTagBFP2 and fluorescence was measured by flow cytometry to quantify the repression efficiency of each gRNA array. (b) Normalized repression of fluorescence by multiplexed expression of gRNA arrays. All experimental samples were normalized to the fluorescence measured for this yeast strain also expressing an array with 3 ‘null’ gRNAs. The ‘3 Null gRNAs’ control was set to a value of 1.0 for all three fluorescence channels. All values plotted are averages from n=8 (3, 6, 9, 12


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gRNA arrays) or n=4 (3 Null gRNAs) samples. Error bars represent one standard deviation from the geometric mean. Asterisks denote two-tail p-value as determined by two-sample ttest, with **p ≤ 0.01, and ***p ≤ 0.0001.

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This assembly method is rapid, inexpensive, and fully compatible with the existing

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Yeast MoClo Toolkit1. It can be used to express at least 12 gRNAs from one promoter,

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which may prove useful in multiplexed CRISPR applications, such as the

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transcriptional regulation of multiple genes in a pathway, or to construct more complex,

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gRNA-based logic circuits13.

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MATERIALS AND METHODS

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Assembly and Validation of gRNA Arrays

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A detailed protocol, including primer sequences and thermocycler settings, can be

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found in the Supporting Information (Supplementary Tables S1-S13). 5 ng of GGV

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template was PCR amplified with a phosphorylated reverse primer and a forward

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primer with 20 nucleotide overhang to add spacer sequences. PCR mixtures were

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then digested with 0.3 µL DpnI at 37°C for at least 30 minutes and the entire mixture

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loaded in an agarose gel for electrophoresis. Fragments corresponding to the

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expected band sizes were excised and purified with a Zymoclean Gel DNA Recovery

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kit (Zymo Research) and then ligated with T4 ligase. A second round of PCR to add

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BsmBI cut sites and four-base overhangs was performed with 10 ng of each ligated

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vector as template. PCR reactions were digested with 0.3 µL of BsmBI and DpnI for

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30 minutes at 37°C and 30 minutes at 55°C, then gel purified again. Golden Gate

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assembly was then performed, with all fragments and the DV diluted to 50 nM before

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use. 0.15 µL DV, 0.5 µL each gRNA part, 1 µl T4 DNA ligase (NEB), 1 µl 10X T4 ligase

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buffer (NEB) and 1.5 µl of BsmBI enzyme (NEB) were added to a 10 µl total reaction

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volume with ultrapure water. Assembly reactions were incubated for thirty cycles, with

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5 min at 37°C and 5 min at 16°C, followed by a 5 min, 55°C final incubation step prior

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to transformation into TurboComp cells (NEB). Transformed cells were plated on LB

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+ chloramphenicol and screened visually for colonies lacking GFP under blue light.

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Colonies lacking GFP upon visual inspection were picked, incubated at 37°C and their

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DNA isolated. Restriction digests were performed with 0.5µl BsaI (NEB) on 100 ng of

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DNA from each colony at 37°C for 1 hour and gel electrophoresis performed to verify



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insert sizes. Of those colonies that yielded DNA bands of expected sizes, DNA from

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three colonies with each array (3, 6, 9, 12) were sent for additional validation by Sanger

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sequencing.

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Construction of Single gRNA Vectors for Quantification of Repression

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To construct gRNAs for targeting dCas9 to one of the three promoters driving the

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expression of a fluorescent protein, we used the pYTK050 part from the Yeast MoClo

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Toolkit1. pYTK050 contains a phenylalanine tRNA, HDV ribozyme, the Cas9/dCas9

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scaffold (sgRNA) sequence and a SNR52 terminator. Complementary oligos were

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ordered (IDT) and annealed, with two additional ‘T’ nucleotides added before the

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spacer sequence to enable its ligation into pYTK050. Annealed oligos were then

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ligated into pYTK050 via BsmBI-mediated Golden Gate assembly. pYTK050 plasmids

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containing designed spacer sequences were genome-integrated at the URA3 locus.

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Strains and Cell Cultures

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All Saccharomyces cerevisiae strains generated in this study are derivatives of

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BY4741 (S288C MATa his3∆1 leu2∆0 met15∆0 ura3∆0) and are listed in

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Supplementary Table S14. All experiments were performed in synthetic complete

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media with 2% (w/v) glucose (VWR), 0.67% (w/v) Yeast Nitrogen Base without amino

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acids (Sigma), 0.14% (w/v) Yeast Synthetic Drop-out Medium supplements without

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histidine, leucine, tryptophan, and uracil (Sigma), 20 mg/L histidine (Sigma), 100 mg/L

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leucine (Sigma), 20 mg/L tryptophan (Sigma), 20 mg/L uracil (Sigma).

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Yeast colonies were inoculated into liquid Synthetic Complete media with all amino

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acids supplemented apart from the corresponding, auxotrophic amino acids (Uracil,

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Leucine and Histidine) and incubated in 96-well, 2.2 mL deep well plates at 30°C and

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700 rpm.

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Flow

Cytometry

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Cell fluorescence was measured by a LSRFortessa X-20 flow cytometer (BD

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Biosciences) using the following settings: FSC 330 V, SSC 250 V, mTagBFP2 - 405

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nm line (450/50) 378 V, sfGFP - 488 nm line (530/30) 450 V, mRuby2 - 640 nm line

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(685/35) 590 V. Fluorescence data was collected from 10000 viable cells for each

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experiment and analyzed using FlowJo software.


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254 255

SUPPORTING INFORMATION

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Detailed protocol of the method to assemble gRNA arrays, including all DNA

257

sequences, primers and parts used in this study, supporting data and additional

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methods.

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ACKNOWLEDGMENTS

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This work was supported by BBSRC grant (BB/R01602X/1) (to RLA), BBSRC grant

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BB/K019791/1 (to TE) and a Fulbright – Imperial College London Partnership award

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(to N.S.M).

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DECLARATION OF INTERESTS

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A patent application related to this work has been filed by the authors.

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REFERENCES

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Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast

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