Systematic Evaluation of CRISPRa and CRISPRi Modalities Enables

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Systematic evaluation of CRISPRa and CRISPRi modalities enables development of a multiplexed, orthogonal gene activation and repression system Andrea Martella, Mike A Firth, Benjamin J. M. Taylor, Anne U. Goeppert, Emanuela M. Cuomo, Robert G. Roth, Alan J. Dickson, and David I. Fisher ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00527 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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ACS Synthetic Biology

Systematic evaluation of CRISPRa and CRISPRi modalities enables development of a multiplexed, orthogonal gene activation and repression system

Andrea Martella1, Mike Firth2, Benjamin J. M. Taylor1, Anne Göppert1, Emanuela M. Cuomo1, Robert G. Roth3, Alan J. Dickson4 and David I. Fisher1* 1. Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK 2. Data Sciences and Quantitative Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK 3. Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden 4. Manchester Institute of Biotechnology, Faculty of Science and Engineering, University of Manchester, M1 7DN, UK

ABSTRACT: The ability to manipulate the expression of mammalian genes using synthetic transcription factors is highly desirable in both fields of basic research and industry

for

diverse

applications,

including

stem

cell

reprogramming

and

differentiation, tissue engineering and drug discovery. Orthogonal CRISPR systems can be used for simultaneous transcriptional upregulation of a subset of target genes whilst downregulating another subset, thus gaining control of gene regulatory networks, signalling pathways and cellular processes whose activity depends on the

ACS Paragon Plus Environment

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expression of multiple genes. We have used a rapid and efficient modular cloning system to build and test in parallel diverse CRISPRa and CRISPRi systems and develop an efficient orthogonal gene regulation system for multiplexed and simultaneous up- and downregulation of endogenous target genes.

KEYWORDS: CRISPRa, CRISPRi, gene regulation, orthogonal CRISPR systems,

DNA assembly, combinatorial assembly, multiplexing

Programmable control of gene expression is indispensable to understanding gene function, identifying new biological mechanisms and developing therapeutics. In particular, simultaneous upregulation of a subset of endogenous genes whilst downregulating another subset in a defined manner raises the possibility of manipulating complex gene networks and multiple cellular processes at once. The CRISPR/Cas9 system has emerged as an exciting platform for engineering synthetic transcription factors to modulate endogenous gene expression

1,2.

Cas9 is

an RNA-guided endonuclease that can be directed to cut a specific locus on doublestranded

DNA

provided

certain

criteria

are

met.

The

process

requires

complementarity between the Cas9-associated guide RNA (gRNA) and the target


2

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site in addition to the presence of a short protospacer-adjacent motif (PAM) 3. Early efforts at engineering the Cas9 protein discovered residues critical to the DNA cutting activity that, when mutated, generate forms of the protein that are still capable of DNA binding but lack detectable nuclease activity

4,5.

These nuclease-

null, or ‘dead’, Cas9 (dCas9) variants can be used as targeting moieties designed to deliver a functional cargo to a specific locus. When combined with transcriptional activators and repressors and targeted to the promoter region of endogenous genes they enable sequence-specific regulation of gene expression 6. dCas9 has been used to target the activation domains of several transcription factors such as p65, HSF1 and MyoD, and viral transactivators such as Rta and VP16, to induce gene expression

5,7-9.

These domains promote the recruitment of chromatin

modifiers, which cause chromatin decondensation and accumulation of histone marks promoting transcription. Similarly, gene repression has been achieved by using dCas9 to target repressor domains such as the Krüppel-associated box (KRAB) that recruits KAP1/TRIM28 and HP1 proteins, hindering the positioning of RNA polymerase II

9-11.

These factors also promote the recruitment of histone

methyltransferases, which increase the levels of tri-methylation of histone H3 at lysine 9 (H3K9me3) causing local compaction of chromatin. Orthogonal systems for simultaneous CRISPR-based gene activation (CRISPRa) and inhibition (CRISPRi) have been developed using dCas9 proteins from different species

12-14.

By associating each Cas9 orthologue to an activator or a repressor,

one can direct the effector domain to the desired genomic site based on the targeting


3


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sequence of its cognate gRNA. Alternatively, multiplexed transcriptional activation and repression has been carried out using gRNA molecules modified to include orthogonally acting aptamers

15-19.

In these cases, each gRNA encodes information

both for DNA target recognition and recruitment of a specific RNA binding protein (RBP). Discrete RBPs can be fused to different effector domains providing for orthogonal complex recruitment. This design allows for simultaneous gene activation and repression within the same cell when orthogonal gRNA aptamers–RBPs are used for different gene targets. In a recent publication, Church’s lab performed a comparative analysis of the different CRISPRa systems to investigate their ability to induce robust gene expression

20.

The same lab has also compared the effectiveness of dCas9 fused

with KRAB and MeCP2 in downregulating the expression of several target genes compared with the use of dCas9 alone 21. To our knowledge, a comprehensive combinatorial comparison of CRISPRa/i systems in the same cellular context aiming to generate an orthogonal system capable of efficient simultaneous up and down-regulation of mammalian endogenous genes is still missing. In the work we present we have assembled a combinatorial library of previously published CRISPRa/i systems to analyse their relative potency and effectiveness in activating or repressing endogenous genes. From the data obtained we have identified an optimised, multiplexed CRISPRa and CRISPRi system exemplified in Expi293F cells. In addition we provide a detailed


4

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framework for the expression and evaluation of CRISPR-based synthetic transcription factors for efficient simultaneous up- and down-regulation of endogenous target genes, providing the community with a validated set of reagents with tremendous potential in both biomedical research and industry.

RESULTS

Modular assembly of CRISPR expression vectors used in the comparative study The cloning of expression vectors encoding for the different CRISPRa/i components and for gRNAs was carried out with a rapid and efficient modular DNA assembly strategy based on a one tube, one step Golden Gate (GG) cloning reaction

22-24

and

a standardized library of compatible genetic parts (Fig.1) (Supplementary Text 1). A DNA element that occupies a module in the platform is interchangeable with alternative DNA parts standardized to occupy the same functional module and thus DNA parts are freely interchangeable, and reusable. The CRISPRa/i expression platform allows the user to perform a combinatorial assembly, parallelizing construction of different versions of the same plasmid differing in promoters, polyA signal sequences or coding sequences (Fig.1B). Although we compared the combinations of different constitutive promoters and several polyA signal sequences, we observed no significant functional change in the cellular context used in our experiments (data not shown). However, we believe having the possibility of using


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alternative promoters (such as inducible promoters) and alternative polyA signals might be desirable in other experimental conditions or cellular contexts. On the other hand, if the choice of promoter and polyA signal is kept unchanged, expression plasmids differing only in the coding sequence will benefit from having identical transcription unit design and identical plasmid DNA backbone. This standardization is an important requirement for a more accurate and reliable comparison study. The modularity of the gRNA cloning platform (Fig.1C) allows for an easily exchange of promoters, crRNA sequences and different trRNA scaffolds leaving the rest of the plasmid unchanged. Together these modular platforms provide a flexible and efficient method for rapid generation of plasmids for the expression of CRISPRa/i proteins and sgRNAs.

Prevalence of PAM sequences for orthogonal gRNA-guided nucleases Orthogonal dCas9 proteins from different bacteria species use distinct PAM sequences and recognise only their cognate gRNA

12.

Thus, expressing multiple

Cas9 orthologues fused with activator and repressor domains in the same cell would enable multiplexed, simultaneous transcriptional activation and repression of different endogenous genes. However, the prevalence of the required PAM sequences in the genome must be considered, as relatively limited flexibility in genome targeting might prevent the use of some gRNA-guided nucleases.


6

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We have considered the genomic window -400 bp to +400 bp from the transcription start site (TSS), considered as a good candidate target region for design of efficient CRISPRa/i gRNAs

10,25-27

and determined the average frequency of PAM sequence

required by different gRNA-guided endonucleases for all protein-coding human genes. We have also focused the analysis on the shorter genomic windows of 400 bp upstream of the TSS, considered to be optimal for the design of CRISPRa gRNA 10

and -50 bp to +50 bp from the TSS as the design of gRNA in proximity of the TSS

can often result in a more efficient downregulation of the gene targeted with CRISPRi

10,26,28.

Among the endonucleases examined, S. pyogenes Cas9 (SpCas9)

and its evolved variant xCas9

29

have a significantly higher frequency of PAMs

compared to other Cas9 endonucleases, which on the contrary show poorer coverage of the genomic window considered (Fig.2). All nucleases other than SpCas9 (and variants) have a number of genes with no PAMs available for CRISPRa/i targeting (Supplementary Table 1). This makes SpCas9, or its less wellstudied variant xCas9, our preferred choice for targeting the TSS of protein-coding genes in human cells.

Comparative study of different CRISPRa systems SpCas9 requires a short and simple PAM sequence making it an optimal choice due to the high frequency of appropriate targeting sites within the human genome. We therefore aimed to develop an efficient CRISPR-based system capable of modulating multiple target genes using a ‘dead’ version of SpCas9 (SpdCas9) and


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harnessing a CRISPR RNA scaffold recruitment system to generate orthogonality for simultaneous transcriptional up- and downregulation. We started with a comparative analysis of different CRISPRa systems. A widely used CRISPR RNA scaffold recruitment system referred to as synergistic activation mediator (SAM) comprises a dCas9-VP64 fusion protein and gRNAs that have been modified with the addition of two ms2 hairpin aptamers. Each ms2 aptamer recruits the viral RNA binding protein (RBP) ms2 capsid protein (MCP) fused to the transcriptional activator domains p65 and HSF1 (PH), to generate the fusion protein MCP-PH 8. The PH domain is expected to work synergistically with VP64 to upregulate target genes. However, indispensable for the generation of a multiplexed, orthogonal system is the removal of the VP64 moiety from SpdCas9 as the dCas9/gRNA-aptamers complex will serve for recruitment of both activators and repressors through the employment of orthogonal RNA-binding proteins/aptamers pairs. We therefore generated and compared two orthogonal CRISPRa RNA scaffold recruitment systems using SpdCas9 or SpdCas9-VP64 and the two RBP/aptamer pairs MCP/ms2 and PCP/pp7 19,30

fusing each RBP with the PH activation domain. We compared the activity of the

two RBP-aptamer systems against two gold-standard activators: SAM and dCas9 fused to the commonly used tripartite activation fusion VP64-p65-Rta (VPR). We also included the first generation CRISPRa fusion dCas9-VP64 in our comparison. We designed and cloned gRNAs targeting the promoters of a set of weakly (ASCL1, TTN, HBG1 and MYOD1) and intermediate-highly (BCL2, XBP1 and MET) expressed genes in Expi293F cells. Previous reports observed an inverse


8

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relationship between basal expression levels of the genes of interest and the relative upregulation, which can be gained by CRISPRa

7,16.

Weakly expressed genes are

easier to upregulate, thus targeting the promoter with one gRNA is generally sufficient to induce strong upregulation. On the contrary, it is more difficult to increase the transcription rate of already highly expressed endogenous genes and the synergistic action of multiple gRNAs per gene may be required. We tested the CRISPRa systems in parallel, transiently co-transfecting the cells with plasmids encoding for CRISPRa proteins along with plasmids encoding for gRNAs (one gRNA per gene for the weakly expressed genes and a pool of 3 gRNAs per gene for the intermediate-highly expressed genes) (Fig.3).

In parallel, for each system we

generated negative controls by transfecting the cells with the CRISPR proteins and a non-targeting gRNA (NT-gRNA) that has no complementary sequence in the human genome. The level of upregulation of target genes was quantified by qPCR 48 hours post-transfection by measuring the fold change of target gene mRNA relative to control cells transfected with an empty vector. We observed no significant variation in gene expression for cells transfected with NT-gRNA for all the CRISPRa systems tested. We observed an overall good and reproducible performance for dCas9-VPR, dCas9/MCP-PH and dCas9/PCP-PH in upregulation of the target genes. As expected, dCas9-VP64 has little or no impact on the expression of target genes when only one gRNA is used. Interestingly, dCas9/PCP-PH shows stronger upregulation of all target genes compared to the other CRISPRa systems tested. Furthermore, the removal of a VP64-fusion from dCas9 generally improves the


9


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activity level of both dCas9/MCP-PH and dCas9/PCP-PH system. We also investigated whether fusing the VPR domain with MCP or PCP would give higher activation of the target genes but on the contrary we observed a lower upregulation level of the target genes compared to samples transfected with MCP or PCP fused to the PH domain (data not shown). From the results obtained we identified dCas9/PCP-PH as the strongest activator and thus the preferred choice for the establishment of the orthogonal CRISPRa/i system based on an RNA scaffold recruitment strategy. The higher performance of dCas9/PCP-PH and the improvement obtained by using dCas9 instead of dCas9-VP64 in aptamer-guided CRISPRa systems was also tested and confirmed in a different human cell line (Supplementary Figure 2). Expi293F are cells grown in suspension and are originally derived from human embryonic kidney cells, we therefore decided to use HCT116, which in contrast to Expi293F cells are adherent and derived from a human colorectal carcinoma. Comparative study of different CRISPRi systems A similar comparative analysis was performed testing different CRISPRi systems. Binding of dCas9 in proximity of the TSS can interfere with transcription by sterically hindering the elongation of RNA polymerase (RNAP) or inhibiting the initial binding of RNAP to the promoter. However, in eukaryotic systems dCas9 alone often achieves only a modest repression of the targeted genes. Further improvement in transcriptional inhibition can be achieved with the addition of repression domains such as the KRAB domain to dCas9

10.

The dCas9–KRAB (dCas9-K) fusion is


10

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considered the gold standard for dCas9-based repression studies in eukaryotic cells. Recently, more potent transcriptional repression was described utilising the synergistic action of KRAB and methyl-CpG binding protein 2 (MeCP2) fused to the C-terminus of dCas9 with the generation of dCas9-KRAB-MeCP2 (dCas9-KM)

21.

Based on our ultimate aim of RNA scaffold recruitment, we fused the repressive fusion of KRAB-MeCP2 to PCP and MCP, generating MCP-KRAB-MeCP2 (MCPKM) and PCP-KRAB-MeCP2 (PCP-KM) respectively. We compared the performance of these aptamer-dependent systems against dCas9, dCas9-K and dCas9-KM. We designed and cloned gRNAs targeting the promoters of a set of endogenous genes (BRCA1, CANX, MAPT, BCL2, XBP1 and CDK4) that are intermediate-highly expressed in Expi293F cells. We tested the CRISPRi systems in parallel by transiently co-transfecting the cells with plasmids encoding for the CRISPRi proteins along with plasmids encoding for gRNAs. BRCA1, CANX and MAPT genes were downregulated transfecting the cells with 1 gRNA per gene. We used already published gRNA sequences designed to target the genomic window of -50 to +50 bp from the TSS

21,25.

XBP1 and CDK4

were downregulated by transfecting the cells with a pool of 3 gRNAs per gene that we designed to target the genomic window of -50 to +50 bp from the TSS. For BCL2 we used the same set of 3 gRNAs we designed for upregulation, targeting the promoter in the genomic window of -20 to -200 bp from the TSS. For each CRISPRi system we generated negative controls by transfecting the cells with a NT-gRNA that has no complementary sequence in the human genome. The level of downregulation


11


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of the target genes was quantified 72 hours post-transfection measuring the fold change of target gene mRNA relative to control cells transfected with an emptyvector. We observed that, for the set of genes/gRNAs tested, adding KRAB to dCas9 (dCas9-K) generally results in minimal or no improvement of the repressive action of dCas9 alone (Fig.4). Three out of the six target genes tested show no significant difference between dCas9-KRAB and dCas9 alone in terms of repression level obtained. For the other three, dCas9-K when compared to dCas9 alone shows a repression improvement of 33% for XBP1, 14% for MAPT, and 4% for BRCA1. However, statistically better repression was obtained when using dCas9-KM confirming the previously published data

21.

dCas9-KM is performing significantly

better compared to dCas9 alone in six out of six genes tested with an average of 21% repression improvement. dCas9/PCP-KM was not better at downregulating the target genes compared to dCas9 alone. However, dCas9/MCP-KM recapitulates the results obtained with dCas9-KM, with an improved repression activity compared to dCas9 alone (Fig.4). In conclusion, dCas9/MCP-KM represents a CRISPRi system with efficiency comparable to dCas9-KM and giving the highest level of repression achieved in this study. The efficiencies of different CRISPRi systems are expected to be cell line dependant in addition to there being gene specific tendencies. Nevertheless we have observed high performance of dCas9/MCP-KM also in HCT 116 cells (Supplementary Figure 3).


12

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Testing the orthogonal RNA scaffold recruitment system for simultaneous up- and down-regulation of target genes From our comparative analysis the RBP-effector/aptamer pairs PCP-PH/pp7 and MCP-KM/ms2 emerged as the best combination for efficient activation and repression of target genes respectively. This allowed us to combine these two systems into an orthogonal CRISPRa/i platform for simultaneous up- and downregulation of multiple target genes in the same cell. We cloned PCP-PH and MCPKM coding sequences into a single plasmid as a bi-cistronic expression cassette under the control of a constitutively active promoter (Fig.5). We also used a hierarchical GG cloning strategy to assemble six gRNA expression cassettes into an all-in-one gRNA expression plasmid (Supplementary Text 2, Supplementary Figure 1). Each gRNA is designed to target a different gene. The gRNAs targeting BRCA1 and CANX have ms2 aptamers for the recruitment of MCP-KM, whereas the four gRNAs targeting ASCL1, TTN and HBG1 have pp7 aptamers for the recruitment of PCP-PH. Expi293F cells were co-transfected with a plasmid encoding dCas9, a second plasmid harbouring the PCP-PH/MCP-KM bi-cistronic cassette and the all-inone plasmid harbouring the six gRNA expression cassettes. The extent of up- and down-regulation of the target genes were quantified 72 hours post-transfection. The co-expression of different sgRNAs with relatively stable stoichiometry in their expression level, along with the expression of dCas9, PCP-PH and MCP-KM leads to simultaneous activation and repression of multiple endogenous genes in the same cell (Fig.5).


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DISCUSSION

Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful tool for targeted genome activation and repression, in addition to its use in genome editing. CRISPRa/i systems are easy to multiplex, highly specific and since they regulate transcription by acting on promoters of endogenous genes their action is exerted in a more physiological cellular context. CRISPRi is preferable to RNAi, which often suffers from low specificity, and CRISPRa to overexpression using cDNAs, where multiplexed gene overexpression comes with significant cloning and transfection burdens in addition to restrictions on isoform processing. Furthermore, CRISPRa/i can also be used to modulate the expression of non-protein coding genes (lncRNA, etc.) and investigate the regulatory functions of enhancers and silencers. Particularly attractive is the possibility of having programmable control over multiple genes with simultaneous transcriptional upregulation and downregulation. This is what endogenous transcription factors normally do, controlling entire gene networks and signalling pathways or multiple cellular processes at once. Different CRISPRa and CRISPRi systems based on dCas9 fusion proteins or CRISPR RNA scaffold recruitment systems have been employed to achieve efficient modulation of gene expression in mammalian cells.


14

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We have provided a comprehensive comparison of these systems aimed at generating an efficient orthogonal CRISPR-based transcriptional activation and repression platform. In this study we have designed a modular DNA assembly system based on GG cloning to easily and rapidly parallelise construction of diverse expression vectors to deliver different CRISPR systems into target cells for a comprehensive comparative study. The modularity of the cloning platform and the interchangeable nature of the DNA parts also allows for a one-step, one-tube combinatorial assembly resulting in different expression vector variants within the same reaction tube, a powerful tool for screening libraries of new functional effector domains. Despite the lack of specific rules for the design of optimal gRNAs for CRISPRa/imediated transcriptional perturbations that are valid for all possible target genes, there is strong evidence from large datasets on the position-specific effects of gRNAs and several design parameters have been elucidated from large-scale studies and considered to be not gene specific. In general, gRNAs targeting the region upstream of the TSS have largely correlated positively with CRISPRamediated gene activation, while positioning dCas9 variants downstream of, or in close proximity to, the TSS negatively impacts gene expression 10,25,26. Positioning of gRNAs relative to the TSS offers a convenient design strategy for both CRISPRi and CRISPRa, though other local steric and regulatory features of eukaryotic promoters might influence the performance of the tested gRNAs. Thus, the screening of multiple gRNAs per gene is often required for efficient targeting and maximal effect.


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Multiplexing experiments aiming to simultaneously perturbate high number of endogenous genes will probably restrict the number of gRNA per gene to one or two. It is therefore critical to be able to screen a number of gRNAs per gene in order to identify a good candidate. The range of sequences recognized by RNA-guided endonucleases is constrained by the need for a specific PAM. A preliminary analysis on the prevalence of PAM sequences in the genomic windows adjacent to the TSS for all human transcripts have shown that SpCas9 offers a significant higher number of PAM sequences compared to alternative RNA-guided endonucleases and thus a higher chance of identifying at least one efficient gRNA for all protein coding sequences in the human genome, making SpCas9 an optimal choice for a multiplexed CRISPRa/i system. Whilst xCas9 offered even greater PAM flexibility, we saw no added benefit to moving to a less well-studied variant. We therefore opted for an RNA scaffold recruitment system based on the RBP/aptamer pairs MCP/ms2 and PCP/pp7 to construct an orthogonal CRISPRa/i system based on SpCas9. The RNA aptamer-based CRISPRa/i systems also provide additional advantages in terms of delivery into the target cells, both in

vitro and in vivo, as these systems consist of components of smaller size compared to multiple dCas9 fusion proteins. This has clear benefits for plasmid-based delivery but may also be of benefit for viral delivery where packaging is affected by size limitations. From our comparative analysis, the dCas9/PCP-PH system exhibits stronger transcriptional upregulation of several target genes when compared with dCas9-VPR


16

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or dCas9/MCP-PH, which are the most widely used systems for CRISPRa studies. The dCas9/MCP-KM system, on the other hand, shows repression activity comparable to a dCas9-KM fusion protein. Several factors may influence the efficiency of up/down-regulation, such as the basal expression level and the chromatin state of the specific target gene. Nevertheless, despite the magnitude of differences between different targeted genes we clearly show that there is a consistent higher efficiency in up-regulation and down-regulation when using dCas9/PCP-PH and dCas9/MCP-KM, respectively, compared to the other systems tested. Based on these results we have built a platform for the co-expression of SpdCas9, PCP-PH and MCP-KM, which allows for an efficient simultaneous upregulation and downregulation of multiple endogenous target genes. In doing so we have also exemplified a relatively straightforward approach to generating and comparing CRISPRa/i systems in parallel.

MATERIALS AND METHODS

Bacterial cell culture Liquid cultures of E.Coli DH5α were grown in LB Medium at 37 °C. Antibiotics used as follows: kanamycin (25 µg/mL) and ampicillin (100 µg/mL). Standardization of genetic parts for Golden Gate cloning


17


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In GG cloning each DNA fragment must be standardized to occupy a specific position/module of the assembly platform. Standardization requires the DNA fragment of interest to be flanked by a convergent pair of type IIS restriction sites (Esp3I for the protein cloning platform and BsaI for the gRNA cloning platform) that define the corresponding fusion sites for the position/module in the platform that the DNA fragment will occupy. Restriction and fusion sites were added to both ends of DNA fragments by PCR (Supplementary Table 2, Supplementary Table 3, Supplementary Table 7). Golden Gate assembly protocol GG reaction mixtures were prepared as follows: 13 fmol of each DNA part (PCR fragments and receiver vector), 5 U of FastDigest Esp3I (Thermo Fisher Scientific) (or 5 U of BsaI-HF (NEB) for the gRNA cloning platform) and 100 U of T4 DNA Ligase (NEB) in NEB ligation buffer with a final reaction volume of 10 μL. The reaction was performed in a thermocycler using the following program: Step 1, 37 °C for 5 min; Step 2, 37 °C for 5 min; Step 3, 16 °C for 10 min; repeat steps 2-3 for 20 cycles; Step 4, 16 °C for 20 min; Step 5, 37 °C for 30 min; Step 6, 75 °C for 6 min; Step 7, 4 °C hold. Subsequently, 2 μL of the resulting reaction was transformed into

E. coli DH5α competent cells. GG reaction conditions reported here were optimized for high assembly efficiency. A detailed protocol for hierarchical cloning of gRNAs is provided in Supplementary Information.


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Bioinformatic assessment of PAM sequences in the promoter regions of human genes GRCh38 transcription start site coordinates and transcript details for all human transcripts

were

obtained

from

EnsEMBL

BioMart

via

(https://www.ensembl.org/info/data/biomart/biomart_perl_api.html).

the

Perl

For

API each

transcript with a support level of 1 or 2, the genomic region 400 bp upstream of the TSS to 400 bp downstream of the TSS was extracted (taking the strand into account). The count of each PAM motif contained in the region was determined using regular expression matching of the PAM and the reverse complement of the PAM (for example "(TTT.|.AAA)" for Cpf1). The genomic windows of 400 bp upstream of the TSS and -50 bp to +50 bp from the TSS were similarly assessed.

Cell culture and transfections Expi293F suspension cells were maintained in shaking culture at 37 °C, 8% CO2, 700 RPM, 90% relative humidity in Expi293 Expression Medium (Thermo Fisher Scientific). Transient transfection of Expi293F cells was performed in 24 deep well plates using reverse transfection. A total of 2.25 µg plasmid DNA was used in each transfection. For transfections comprising 3 vectors (sgRNA plasmid, dCas9 plasmid and RBP-effector plasmids) a ratio of 2:1:1 was used (respectively). For all 2 vectors systems, an equivalent quantity of empty vector was substituted to ensure equal amounts of DNA were transfected. For experiments involving multiple guides, the


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amount of each gRNA plasmid was adjusted pro rata. For all gRNA spacer sequences (crRNA), please see Supplementary Table 4. The DNA mix was diluted in 75 µl of Expi293 Expression Medium. For each transfection, 10 µg of PEI MAX 40K (Polysciences) was diluted in 75 µl of Expi293 Expression Medium. Diluted PEI MAX 40K was added to each DNA aliquot and incubated for 15 minutes at room temperature. After complex formation, 1.5 ml of culture at 3.5-4.2 x 106 cells/ml was added to each well and incubated at 37 °C, 8% CO2, 700 RPM, 90% relative humidity for 24 hours. At 24 hours post-transfection a further 1.5 ml Expi293 Expression medium was added to each well. HCT 116 (ATCC® CCL-247™) cells were maintained in McCoy’s Modified 5A Medium with 10% FCS at 37 °C, 5% CO2. Transient transfection of HCT 116 cells was performed in 24 well plates using FuGENE® HD Transfection Reagent (Promega) following manufacturer's instructions. A total of 500 ng of DNA/well, with a FuGENE®:DNA ratio of 3:1, was used. For transfections comprising 3 vectors (sgRNA plasmid, dCas9 plasmid and RBP-effector plasmids) a ratio of 2:1:1 was used (respectively). For all 2 vectors systems, an equivalent quantity of empty vector was substituted to ensure equal amounts of DNA were transfected. For experiments involving multiple guides, the amount of each gRNA plasmid was adjusted pro rata.

RNA extraction and qPCR analysis Cells were harvested 48h or 72h post-transfection by centrifugation. RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) and quantified by absorbance at


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A260/280. cDNA was synthesized using the GoScript™ Reverse Transcriptase Kit (Promega) using 1500 ng of RNA per cDNA reaction. qPCR reactions were prepared using either PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) or TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific). 1 μl of 1:10 cDNA dilution was used per reaction in a 10 μl total reaction volume. For PowerUP SYBR Green reactions, cycling conditions were as follows: 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. The latter two steps were cycled for 50 repeats with plate reads taken after the 60 °C step. For TaqMan™ reactions, cycling conditions were as follows: 50° for 2 min, 95° for 10 min, 95° for 15 sec and 60° for 1 min. The latter two steps cycled for 40 repeats with plate reads taken after the 60° step. For qPCR primers/probes, please see Supplementary Table 5. Gene expression was normalized to the expression of the gene GAPDH and HBMS as internal housekeeping genes

ABBREVIATIONS

CRISPRa : CRISPR activation CRISPRi : CRISPR inhibition dCas9-K: dCas9-KRAB GG: Golden Gate KM: KRAB-MeCP2


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MCP: ms2 capsid protein NT-gRNA: Non-targeting gRNA PCP: pp7 capsid protein PH: p65-HSF1 RBP: RNA binding protein TSS: Transcription starting site VPR: VP64-p65-Rta

ASSOCIATED CONTENT Supporting information Description of the modular cloning platforms for the assembly of CRISPR expression vectors used in this work. Description of the strategy used to clone multiple gRNA expression cassettes in an all-in-one plasmid. Additional figures and tables as described in the text. Sequences of the DNA fragments used in this work. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author DIF: Tel: +441625234454; Email: [email protected]


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Author Contributions DIF, BJMT, AUG, EMC, AJD, RGR and AM conceived the study. AM designed and performed the experiments; MF performed the bioinformatic analysis; AM and DIF wrote the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors thank Giovanni Ciotta for assistance in molecular biology and all members of AstraZeneca’s Cell Biology Team for their valuable advice.

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FIGURES Fig. 1 Modular cloning platform for the generation of CRISPRa/i expression vectors A) Schematic representation of the GG assembly for the generation of mammalian expression vectors. Standardized DNA parts are flanked by a pair of Type IIS recognition sites, which generate user-defined 4 bp overhangs (fusion sites) upon cleavage. Parts that belong to the same functional module (and therefore occupy the same position in the platform) are flanked by the same pair of fusion sites (here labelled A-E). Parts from the library are combined with a receiver vector, the appropriate Type IIS enzyme and T4 DNA ligase. During the GG reaction parts are released from their donor plasmids and assembled into the receiver plasmid backbone in a single reaction mix. The yellow arrows indicate the restriction sites. B) Schematic representation of the modular platform used for assembly of plasmids for the expression of proteins. One functional module has been assigned to each position in the platform; the letters on both sides of the modules indicate the flanking 4 bp fusion sites labelled with letters (A-E). Genetic parts are interchangeable and reusable allowing for combinatorial assembly. A=TAGG, B=CAGC, C=AGGC, D=GGTA, E=ACGA. C) Schematic representation of the modular platform used for assembly of plasmids for the expression of gRNAs. One functional module has been assigned to each position in the platform. F=TGCC, G=ACCG, H=GTTT, I=CAGA.

Fig. 2 Frequency of PAMs in the promoter region of human endogenous genes


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Average number of PAMs for the different RNA-guided endonucleases SpCas9

31,

xCas9

32,

29,

StCas9 38

SpCas9 EQR variant 32, SpCas9 VQR variant

33,

NmCas9

34,

SaCas9

and As/LbCpf1 RVR variants

35, 38

FnCpf1

36,

32,

As/LbCpf1

SpCas9 VRER variant

37,

As/LbCpf1 RR variants

in the genomic window of -400bp to + 400bp from

the TSS, -400bp to 0 bp from the TSS and -50bp to +50bp from the TSS for all human transcripts.

Fig. 3 Comparison of different CRISPRa systems A-B) Schematic representation of gRNA designs with or without the addition of ms2 and pp7 aptamers and the CRISPRa proteins used in the comparative study. C) RNA expression analysis of seven endogenous human genes. Data are expressed as fold increases relative to the negative empty vector control. Data are shown as the mean ± s.e.m. (n = 3 independent experiments). P values were determined by a two-tailed t-test compared to control, *P

Systematic Evaluation of CRISPRa and CRISPRi Modalities Enables

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