Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive

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Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive DNA Labeling and Endogenous Gene Regulation Shipeng Shao, Lei Chang, Yuao Sun, Yingping Hou, Xiaoying Fan, and Yujie Sun ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00268 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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ACS Synthetic Biology

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Multiplexed sgRNA Expression Allows Versatile Single Non-

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repetitive DNA Labeling and Endogenous Gene Regulation

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Shipeng Shao1, Lei Chang1, Yuao Sun1, Yingping Hou 1, Xiaoying Fan1, Yujie Sun1,*

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1

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(BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.

State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center

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*E-mail: [email protected]

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*Phone#: 86-10-62744060

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ACS Paragon Plus Environment


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ABSTRACT：：

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The CRISPR/Cas9 system has made significant contribution to genome

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editing, gene regulation and chromatin studies in recent years. High-throughput

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and systematic investigations into the multiplexed biological systems require

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simultaneous expression and coordinated functioning of multiple sgRNAs.

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However, current co-transfection based sgRNA co-expression systems remain

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inefficient and virus-based transfection approaches are relatively costly and labor

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intensive. Here we established a vector-independent method allowing multiple

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sgRNA expression cassettes to be assembled in series into a single plasmid. This

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synthetic biology-based strategy excels in its efficiency, controllability and

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scalability. Taking the flexibility advantage of this all-in-one sgRNA expressing

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system, we further explored its applications in single non-repetitive genomic locus

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imaging as well as coordinated gene regulation in live cells. With its full potency,

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our method will facilitate the research in understanding genome structure,

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function and dynamics.

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Keywords: CRISPR/Cas9, Single-guide RNA, Chromosomal dynamics, Single

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non-repetitive gene, Golden Gate cloning, Transcription regulation

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INTRODUCTION

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Since its discovery, the clustered regularly inter-spaced short palindromic

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repeats (CRISPR)-associated Cas9 nuclease has largely revolutionized the fields

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in genome editing, gene regulation and chromatin studies in live cells

32

Compared to protein-based recognition systems such as ZFN and TALE,

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CRISPR recognizes target DNA via Watson-Crick base pairing, allowing it to

34

perform high-throughput chromatin targeting in an effective and efficient way 2.

35

Emerging new versions of the CRISPR/Cas9 system have been adopted to

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expand our capacity for precise genome manipulation

37

carry tremendous potentials in both biomedical researches and therapeutic

38

genomic targeting 8.

39

1

.

3-7

. These powerful toolkits

Simultaneous expression and coordinated functioning of multiple sgRNAs in 9

40

live cells are often necessary in circumstances such as simultaneous knockout

41

and regulation of multiple endogenous genes 10. Notably, CRISPR-based labeling

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and imaging of genomic loci with non-repetitive sequences in live cells also

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require to tile the loci with multiple sgRNAs 11.

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There are several approaches available for simultaneous delivery of multiple

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sgRNAs to scale up the targeting capacity of the CRISPR/Cas9 system. For

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instance, cells can be transfected with a mixture of multiple sgRNA plasmids by

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chemi-transfection or electro-transfection

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transfection of different plasmids is relatively inefficient and also results in

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variable expression levels of each sgRNA in single cells. Similarly, delivery of

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multiple sgRNAs using lentivirus infection

12, 13

. However, such transient co-

11

can also lead to variable efficacy in

3



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delivery and expression. Alternatively, although microinjection can deliver multiple

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sgRNAs into a single cell without the limitation of plasmid type and number

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suffers from low throughput and requires special equipment and superb skills.

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With the evolution of synthetic biology, new cloning methods have emerged in the

55

last two years to achieve high speed, efficiency and flexibility in assembly of

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multiple DNA fragments

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used for fast assembly of TALE repeats

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sgRNAs

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reaction is quite limited following current protocols as the assembly efficiency

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decreases dramatically as the number of cassettes increases. Even though a

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recently developed hierarchical assembly method could assemble multiple

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sgRNAs, as it relies on receptor plasmids for every step, the whole procedure

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requires several rounds of transformation, verification, plasmid extraction, and

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thus on average takes more than 20 days to assembly 20 sgRNA expression

65

cassettes into a single plasmid

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sgRNA expression system needs to balance in ease of implementation, delivery

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efficiency, precise stoichiometry control, and cost of time.

14

, it

15-17

. For instance, the Golden Gate cloning method was 18

and for assembly of short serial

9, 19

. However, the number of sgRNA cassettes assembled in one

20

. Collectively, a well-rounded multiplexing

68

In this work, we have developed a hierarchical cloning strategy to assemble

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multiple sgRNA expression cassettes into a single vector. We demonstrated that

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this new approach is not only robust and time-saving in assembly of different

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numbers of sgRNA expression cassettes at will but also able to assure precise

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control of stoichiometry of different sgRNAs. Using this all-in-one sgRNA

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expressing system, we successfully labeled and imaged non-repetitive genomic

4


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loci, MUC4 and HER2 genes, in living human cells. Additionally, we demonstrated

75

that the all-in-one sgRNA expressing system is highly efficient in gene activation

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and repression. Using multiple sgRNAs to tile the promoter of endogenous gene

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SOX2 to recruit multiple transcriptional regulators, we found that the regulation

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efficiency by this synergistic gene regulation approach was superior to that by

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single sgRNA and co-transfection of multiple sgRNAs. We also showed that the

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all-in-one sgRNA expressing system allows parallel expression of different

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sgRNAs with relatively stable stoichiometry in their expression level, leading to

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reliable simultaneous activation and/or repression of multiple endogenous genes

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in the same cell. In summary, our modular sgRNA assembly system can be

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readily automated and will be highly useful for applications in cell biology and

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genetics, such as gene editing, gene regulation, pathway engineering and live cell

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chromatin labeling.

87

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RESULTS

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Plasmid-independent hierarchical assembly of multiple sgRNA expression

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cassettes

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To achieve high-throughput assembly of multiple sgRNA cassettes, here we

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have developed a plasmid-independent, PCR-based hierarchical assembly

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strategy. Using a series of three one-pot Golden Gate cloning with type II

94

restriction endonuclease, we demonstrated the robustness and high efficiency of

95

our method in assembly of repeated units (Figure 1A). Firstly, PCR-amplified

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conventional or functionally modified sgRNA scaffolds and U6 promoter were

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ligated with respectively synthesized targeting oligos by Golden Gate cloning.

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Then the reaction mix was amplified by PCR with U6-forward and sgRNA-

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scaffold-reverse primers without purification (Figure 1B). Thirdly, we designed

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junctions allowing ordered ligation to form the intermediate fragments. We used

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these junctions to assemble five sgRNA cassettes into one by a second round of

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Golden Gate cloning (Figure 1C). Lastly, a third Golden Gate reaction was

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performed to assemble the five-cassette-intermediates into the final construct

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carrying all 20 sgRNAs. The whole assembly procedure takes about 4-5 days.

105

Random selection of end clones were verified (Figure 1D). Aside from high-

106

efficiency, high-speed and high-throughput in generating a multi-sgRNA cluster,

107

our approach also provides the flexibility for assembling different numbers of

108

sgRNAs using varied combinations of the forward and reverse primers. To see

109

whether the multiplexing sgRNA expression system will influence the expression

110

level of each sgRNA, we performed RT-PCR to quantify the expression level of

6


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individual sgRNA (Supplementary Figure 1). The results indicated that all sgRNAs

112

were expressed at a similar expression level.

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Establishment of a stable cell line expressing the SunTag system

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We fused the SunTag signal amplification system

21

to dCas9 for the

115

enhancement of fluorescence signal that one sgRNA can recruit. A previous

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study has shown that telomeres labeled with dCas9-SunTag were nearly 20-fold

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brighter compared to those labeled by dCas9 without alteration of the telomere

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mobility

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SunTag has not been realized. We reasoned that with dCas9-SunTag,

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simultaneous expression of multiple sgRNAs in a single cell can realize non-

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repetitive chromosomal locus labeling with high quality and efficiency (Figure 2A).

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Transient expression of two components of the SunTag system usually

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results in either incomplete labeling or excessive background from unbound

124

molecules. In order to minimize those effects, we integrated the SunTag

125

expression cassette into the genome using the PiggyBac transposon system

126

and generated a stable cell line with minimal expression level of dCas9-

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(GCN4)X24-P2A-BFP and scFV-sfGFP-GB1 (Figure 2B and Supplementary

128

Figures 2 & 3). With the low expression level, diffusing dCas9 single molecules

129

were able to be observed in the GFP channel (Supplementary Figure 4A) and

130

repetitive chromosomal loci, such as telomeres or the 2rd exon of MUC4 gene,

131

were successfully labeled with dCas9-SunTag in live MDA-MB-231 cells

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(Supplementary Figure 4B & C). In order to compare the signal-to-noise ratio

133

(SNR) between dCas9-GFP and the dCas9-SunTag system, the tandem array of

21

. However, labeling of non-repetitive chromosomal loci using dCas9-

7


22


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5S ribosomal DNA (5S rDNA) on chromosome 1, which consists of 17 repeats,

135

was labeled and quantified (Supplementary Figure 5). The results showed that

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the SNR was improved nearly 6 folds with dCas9-SunTag. The significant

137

improvement in SNR allows imaging with low laser power and short exposure

138

time, enabling long-term tracking of chromosomal loci.

139

Single non-repetitive sequence labeling by multiplexed sgRNA expression

140

We next tried the multiple sgRNA expressing strategy for non-repetitive

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sequence labeling in living cells. We first chose MUC4 gene as a demonstration

142

as it has tandem repeat regions advantageous for the validation by FISH.

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Specifically, 20 sgRNAs were deigned against a stretch of DNA (about 1.5 kb)

144

and cloned into one plasmid by the hierarchical assembly method. The SunTag

145

cell line was transfected with the all-in-one plasmid and imaged 24 h after

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transfection.

147

To verify the specificity and efficiency of non-repetitive MUC4 gene labeling,

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we performed two-color imaging with CRISPR probes targeting the non-repetitive

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sequence in the first intron of MUC4 and FISH probes targeting the polymorphic

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tandem repeats in the third exon. Because the harsh conditions of FISH sample

151

preparation disrupted the fluorescence signal of GFP, we performed immuno-

152

fluorescence against GFP. Colocalization of the two channels indicated that the

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CRISPR probes indeed labeled the MUC4 locus (Figure 2C). In order to quantify

154

the robustness of our method, we also measured the cell labeling efficiency and

155

loci number per cell (Supplementary Figure 6).

8


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By labeling specific non-repetitive genomic loci with CRISPR, we could

157

investigate chromosome reorganization at different cell cycle stages (Figure 2D &

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Supplementary Figure 7). Similar results were also obtained for another non-

159

repetitive gene HER2 (Supplementary Figure 8). These results indicate that

160

combination of the all-in-one sgRNA expression plasmid and SunTag signal

161

amplification system can be used to label arbitrary sequences in the genome,

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visualize their positions in the nucleus, and study the relationship between the

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gene position and its expression 23.

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We further tracked the dynamics of the MUC4 locus labeled with dCas9-

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SugTag (Supplementary Movie 1). Two typical trajectories in the same cell were

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observed (Figure 3A). No obvious correlation was found between the two allelic

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loci, indicating the heterogeneity of the surroundings of allelic genes. We

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calculated the average mean square displacement of 17 different loci and

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extracted the anomalous diffusion scaling exponents (α = 0.34) (Figure 3B),

170

indicating that the movement of chromosomal loci is subdiffusive. We also ruled

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out the possibility that the observed movement of chromatin locus was caused by

172

localization error (Supplementary Figure 9) or stage drift (Supplementary Figure

173

10).

174

To obtain a more robust measurement of the subdiffusive behavior of the δ v(τ).

175

MUC4 locus, we calculated the average velocity autocorrelation function C

176

This function describes what degree the average velocity over a time interval δ is

177

correlated with the average velocity over another time interval δ that is separated

178

by τ from the first one

24

. The correlation function C

9


δ v(τ)

was calculated for


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δ v(τ)

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different values of δ and plotted as a function of the time lag τ. For all δ, C

180

dropped into negative values before finally decaying to zero, indicating the

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negative correlation of MUC4 loci (Figure 3C, left). We then analyzed the

182

behavior of C

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shown (Figure 3C, right). This indicates that the motion of MUC4 segments

184

possessed the same negative correlation property at different temporal scales.

185

Taken together, the results indicate that the MUC4 locus underwent a ‘back and

186

forward’ movement, i.e. motion in one direction is likely to be followed by motion

187

in the opposite direction, which may arise from restoring forces of its surrounding

188

chromatin fiber.

189

Synergistic regulation of endogenous genes by multiplexed sgRNA

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expression

δ v(τ)

as a function of the ratio (τ/δ), and a single master curve was

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Besides the single non-repetitive DNA sequence imaging, the multiplexed

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sgRNA expression strategy can also be used for synergistic regulation of

193

endogenous genes as multiple sgRNAs can recruit multiple copies of proteins to

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function synergistically. We designed sgRNAs targeting the human SOX2 gene in

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HEK293T cells (Figure 4A). We found that the efficiency of synergistic activation

196

of endogenous genes was much higher than that of single sgRNA-mediated

197

activation or cotransfection of multiple sgRNAs. Furthermore, similar results were

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obtained for Cas9-KRAB mediated SOX2 repression experiments (Figure 4B).

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Coordinated activation and repression of endogenous genes by multiplexed

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sgRNA

10


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To examine the capability of coordinated gene regulation of our multiplexed

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sgRNA expression method, we designed 20 sgRNAs each targeting a randomly

203

chosen gene. HEK293T cells were transfected with the all-in-one sgRNA

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expressing plasmid with dCas9-SunTag-VP64 or dCas9-KRAB for activation or

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repression, respectively. The results indicate that all 20 randomly chosen genes

206

were up-regulated or down-regulated simultaneously (Supplementary Figure 11 &

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Supplementary Figure 12).

208

At last, we tested the all-in-one plasmid for simultaneous activation and

209

repression of different genes in the same cell. Simultaneous up-regulation of a

210

subset of endogenous genes while down-regulation of another subset of

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endogenous genes in a defined manner can open up novel opportunities for

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systems biology as it raises the possibility of manipulating transcriptional

213

networks. We used functional modified sgRNA for this application as different

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sgRNAs with different aptamers insertion can recruit different transcriptional

215

regulators (Figure 5A). The sgRNAs targeting the first group of 10 genes were

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inserted with PP7 at their tetraloop and loop2, while sgRNAs targeting the second

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group of 10 genes were inserted with MS2 at the same position 6. With co-

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transfection of tdPCP-VPR

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expressing plasmid, regulation of different genes was as expected and the

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regulation result was orthogonal for the corresponding genes (Figure 5B). These

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results indicate that the multiplexed sgRNA expression strategy can be expanded

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with modified sgRNA for multi-function applications.

25

, tdMCP-KRAB, dCas9, and the all-in-one sgRNA

223

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DISCUSSION

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CRISPR based technologies have revolutionized the fields of genome editing

226

and gene regulation. Simultaneous expression and coordinated functioning of

227

multiple sgRNAs will add a new dimension to the evolving CRISPR system,

228

further facilitating our understanding of the physiological and pathological

229

conditions.

230

Here we have reported a hierarchical assembly strategy for rapid assembly

231

of multiple sgRNA expression cassettes into a single vector. Compared to the

232

current methods, this system ensures high speed, efficiency, capacity and

233

flexibility. Based on Golden Gate cloning, our hierarchical assembly strategy only

234

takes 4-5 days to assemble 20 sgRNA expression cassettes into one plasmid with

235

high fidelity.

236

Moreover, the hierarchical assembly strategy also offers the flexibility in

237

tuning the number of sgRNAs assembled simply by choosing different

238

combination of amplification primers. Meanwhile, the expression level of different

239

sgRNAs in the all-in-one vector could also be differentiated by using different

240

promoters

241

sgRNA expression system can also be used in expressing sgRNAs for other

242

orthogonal CRISPR systems, including SA Cas9

243

single all-in-one vector can be used for multiplexing operations on the same

244

genomic region, such as imaging, transactivation, repression and chromatin

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epigenetic modification 29.

19

. Additionally, besides the SP CRISPR system, the multiplexed

26

, Cpf1

12


27

and C2c2

28

. Lastly, a

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In this work, we have demonstrated assembly of 20 sgRNAs in one single

247

vector. Regarding the capacity of the all-in-one sgRNA expression vector, as the

248

upper limit of the sgRNA number that can be assembled is determined by the

249

carrying capacity of the plasmid, there are several possible ways to enhance the

250

system. Firstly, generating multiple sgRNAs from mRNA

251

sgRNAs

252

needed for the transcription of multiple sgRNAs. Secondly, since the longest

253

fragment in our all-in-one construct is U6 promoter, using other expressing

254

systems will further increase the capacity of our cloning methods. With the

255

improvement of the DNA assembly methods, it is possible that a library of

256

sgRNAs targeting a long DNA sequence can be assembled into one vector, which

257

will greatly aid on the study of structure and dynamics of whole chromosomes or

258

large chromatin domains in living cells. So far, we have only demonstrated the

259

functionality of our all-in-one multiple sgRNA expression vectors in cultured cells.

260

Due to the excessive length and repetition of the multiple sgRNA expression

261

vector, it would be quite challenging to pack it in virus and apply it in in vivo

262

studies.

263

METHODS

264

Hierarchical assembly of multiple sgRNA expression cassettes

31

30

or tRNA-flanked

can make the construct much shorter because only one promoter is

265

All primers were ordered from Ruibo Biotech (Beijing). The U6 promoter,

266

conventional and modified sgRNA expression cassettes were amplified from

267

plasmids

268

psgRNA2.02-MS2-ccdB (Addgene #: 99911, Supplementary Map 2), and

psgRNA-ccdB

(Addgene

#:

99915,

Supplementary

13


Map

1),


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269

psgRNA2.02-PP7-ccdB (Addgene #: 99912, Supplementary Map 3). The oligos

270

(F + R, 100 µM each) for each targeting sgRNA were phosphorylated by T4 PNK

271

(NEB) and annealed in a thermocycler by using the following parameters: 37 °C

272

for 30 min; 95 °C for 5 min; ramp down to 25 °C at 5 °C min-1. The

273

phosphorylated and annealed oligos were then diluted to 1:100 by adding 1 µl

274

oligos to 99 µl ddH2O.

275

For the first assembly step, U6 promoter (10 ng), annealed oligos (1 µM) and

276

sgRNA scaffold (5 ng) were mixed with BsmBI (1 µl, NEB) and T4 ligase (0.5 µl,

277

NEB) together for Golden Gate reaction in 20 separate tubes by using the

278

following parameters: 37 °C for 10 min; 16 °C for 10 min; 10 cycle; 55 °C for 10

279

min; 85 °C for 10 min. The reaction mix was then diluted by 200 folds in ddH2O. 1

280

µl diluted mix was used as template for PCR reaction. The primer sequence and

281

allocation have been described in Supplementary Tables 1 and 2. After the PCR

282

reaction, gel electrophoresis was used to check the reaction. After gel extraction,

283

a QIAquick gel extraction kit was used to ensure that the product contains only

284

the intended U6+oligos+sgRNA ligation product.

285

For the second assembly step, the 20 ligation fragments (10 ng each) were

286

mixed in 4 separate tubes (1-5 in tube #1, 6-10 in tube #2, 11-15 in tube #3, 16-

287

20 in tube #4) with BsaI (1 µl, NEB) and T4 ligase (0.5 µl, NEB) together for

288

Golden Gate reaction by using the following parameters: 37 °C for 10 min; 16 °C

289

for 10 min; 10 cycle; 37 °C for 10 min; 85 °C for 10 min. The reaction mix was

290

then diluted by 200 folds in ddH2O. 1 µl diluted mix was used as template for the

291

PCR reaction. The primer sequences and allocation were described in

14


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292

Supplementary Tables 1 and 2. After the PCR reaction, gel electrophoresis was

293

used to check the reaction. After gel extraction, a QIAquick gel extraction kit was

294

used to ensure that the product contains only the intended 5 ligation length

295

product. This PCR step needs to be optimized to ensure the maximum

296

productivity due to the repetitive sequence of the 5 sgRNA expression cassettes.

297

If the intermediates (5 sgRNA expression plasmids) were needed, the Golden

298

Gate reaction should be performed with the corresponding receptor plasmids.

299

For the third assembly step, the 4 ligated fragments (100 ng each) and 50 ng

300

receptor plasmid pMulti-sgRNA-LacZα (Addegene #: 99913, Supplementary Map

301

4) or pMulti-sgRNA-LacZα-DsRed (Addegene #: 99914, Supplementary Map 5)

302

were mixed with BpiI (1 µl, Thermo Fisher Scientific) and T4 ligase (0.5 µl, NEB)

303

together for Golden Gate reaction by using the following parameters: 37 °C for 10

304

min; 16 °C for 10 min; 10 cycle; 37 °C for 10 min; 85 °C for 10 min. The

305

competent E. coli strain Stbl3 (TransGen Biotech) was transformed with the final

306

reaction mix and cultured in 30 °C to minimize the opportunity of losing the repeat

307

units. The plasmids were linearized and checked by gel electrophoresis to ensure

308

that the final plasmid contains the proper length of repeats. The more detailed

309

information about the protocol to assemble 20 sgRNAs was included in

310

Supplementary Information as Supplementary Protocol 1.

311

Construction of other plasmids used in this work

312

The NLSSV40-dCas9-(NLSSV40)X3-(GCN4-V4)X24-NLSSV40-P2A-BFP fragment

313

was amplified by PCR from plasmid pHRdSV40-NLS-dCas9-(GCN4-V4)X24-NLS-

314

P2A-BFP-dWPRE (Addgene #: 60910) and then ligated into PiggyBac plasmid

15



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(home-made)

Page 16 of 32

315

pB-TRE3G-BsmBI-EF1α-PuroR-P2A-rtTA

by

Golden

Gate

316

assembly with BsmBI and T4 ligase (NEB). The scFV-sfGFP-GB1-NLSSV40

317

fragment was amplified from plasmid pHR-scFV-GCN4-sfGFP-GB1-NLS-dWPRE

318

(Addgene #: 60906) and then ligated into PiggyBac plasmid pB-TRE3G-BsmBI-

319

EF1α-HygroR-P2A-rtTA (home-made) by Golden Gate assembly with BsmBI and

320

T4 ligase (NEB). The tdMCP fragment was amplified from phage-ubc-nls-ha-

321

tdMCP-gfp (Addgene #: 40649), and the KRAB was amplified from pHAGE-EF1α-

322

dCas9-KRAB (Addgene #: 40649) and then ligated into plasmid pB-CMV (home-

323

made). The tdPCP fragment was amplified from phage-ubc-nls-ha-tdPCP-gfp (A

324

Addgene #: 40650), and the VPR was amplified from SP-dCas9-VPR (Addgene

325

#: 63798) and then ligated into plasmid pB-CMV (home-made). The telomere,

326

rDNA10, MUC4-E2 targeting sgRNAs and other sgRNA expression plasmids

327

were made according to procedures described previously 6. All primers and

328

sequences used in this work were listed in Supplementary Tables 1-9.

329

Cell Culture, Transfection, Stable Cell Line Construction

330

Human breast cancer cell line MDA-MB-231 and HEK293T were maintained

331

in Dulbecco’s modified Eagle medium (DMEM) with high glucose (Lifetech), 10%

332

FBS (Lifetech), 1X penicillin/streptomycin (Lifetech). All cells were maintained at

333

37 °C and 5% CO2 in a humidified incubator. All plasmids were transiently

334

transfected with Lipofectamine 2000 (Lifetech) in accordance with the

335

manufacturer’s protocol. To construct a stable cell line, MDA-MB-231 cells were

336

spread onto a 6-well plate one day before transfection. On the next day, the cells

337

were transfected with 500 ng pB-dCas9-(GCN4)X24, 500 ng pB-scFV-sfGFP, and

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338

200 ng pCAG-hyPBase using Lipo 2000. Hygromycin (200 µg/ml) and puromycin

339

(5 µg/ml) were used for the selection of positive clone for about 2 weeks. Cells

340

with proper expression level of dCas9-(GCN4)X24 and scFV-sfGFP were selected

341

using fluorescence-activated cell sorting analysis (FACS). Cells were trypsinized

342

and centrifuged at 300xg for 2 min at RT. The supernatant was removed, and the

343

cells were resuspended in 1× PBS. Fortessa flow analyzers (BD Biosciences)

344

were used for FACS with the following settings. BFP was measured using a 405

345

nm laser and a 450/50 filter and sfGFP was measured using a 488 nm laser and

346

a 530/30 filter. The FACS results were analyzed by FlowJo.

347

To choose a “minimal expression level” of dCas9-SunTag stable clone, a

348

window (as indicated by the yellow window in Supplementary Figure 2A) which is

349

far lower than the averaged transient expression level (several hundred times

350

lower) and slightly higher than the untransfected cells was applied while gating

351

the cells. After the expansion of each clone, the expression level should be

352

compared with the untransfected cells (Supplementary Figure 2B). A locus with

353

low repetitive sequence or non-repetitive sequence was labeled to quantified the

354

labeling results. Based on the labeling performance, optimization can be achieved

355

by choosing a fluorescence intensity gradient. The clone with the best

356

performance was chosen as the stable cell line in the following experiments.

357

FISH and Immunofluorescence

358

The FISH probe targeting the third exon of MUC4 was directly synthesized

359

by Invitrogen with Cy5 labeled at the 5’ end. The sequence of the FISH probe

360

was as following: 5’-Cy5-CTTCCTGTCACCGAC-3’. The transfected cells were 17



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361

washed with PBS briefly once and incubated with extraction buffer (0.1 M PIPS, 1

362

mM EGTA, 1 mM MgCl2, 0.2% TritonX-100) for 1 min to extract cytoplasm

363

diffusive proteins. Cells were then washed once with PBS and fixed with 4% PFA

364

for 15 minutes. Cells were permeabilized with 0.1% saponin/0.1% Triton X-100 in

365

PBS for 10 min at room temperature (RT) and then washed twice in PBS for 5

366

min at RT. Cells were incubated for 1 h in 20% glycerol/ PBS at RT and then

367

frozen/ thawed 3 times in liquid nitrogen. Cells were washed twice in PBS for 5

368

min at RT, incubated in 0.1 M HCl for 30 min at RT and then washed in PBS for 5

369

min at RT. Cells were permeabilized with 0.5% saponin/0.5% Triton X-100 in PBS

370

for 30 min at RT and washed twice in PBS for 5 min at RT. Cells were

371

equilibrated in 50% formamide/2x SSC for 20 min at RT. The probe was added in

372

hybridization buffer and the dish was warmed to 78 °C for precisely 2 min. Cells

373

were incubated overnight at 37 °C in a dark humidified chamber. The next day,

374

cells were washed in the following buffer sequentially, 50% formamide/2X SSC

375

for 15 min at 45 °C, 0.2X SSC for 15 min at 63 °C, 2X SSC for 5 min at 45 °C.

376

and 2X SSC for 5 min at RT.

377

For immunofluorescence, cells were incubated in 0.5% Triton followed by 30

378

min 5% BSA blocking at RT. The cells were then stained with human anti-GFP

379

antibodies (proteintech #66002-1-Ig) in blocking buffer for 60 min, washed with

380

PBS, and then stained with Cy3-labeled secondary antibodies in blocking buffer

381

for 1 h. The labeled cells were washed with PBS, post-fixed with 4% PFA for 10

382

min at RT. Lastly, the cells were stained with DAPI (5 µg/ml in PBS) for 2 min in

383

at RT and then wash 3 times with PBS.

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384 385


qRT–PCR About 48 h post transfection in six-well plates with proper amount of plasmids,

386

total RNA were extracted by TRIzol (Invitrogen). Complementary DNAs (cDNAs)

387

were synthesized using RevertAid First Strand cDNA synthesis Kit (Thermo

388

Fisher Scientific) and qPCR was performed using SYBR Green PCR Master Mix

389

(Roche). GADPH was used as the internal reference. All primers used in the

390

qPCR step were designed by primer-BLSAT on NCBI website. We used the

391

“semi-quantitative PCR” to analysis the data. Briefly, in each group (experimental

392

and control), the GADPH and the gene to be examined (Sample) were amplified

393

using corresponding primers in two different tubes with the same genomic DNA

394

input (10 ng). Each group has three biological replicates. We performed a two-

395

step qRT- PCR to quantify the expression level of the genes of interest. The

396

program is comprised of two reactions: one reaction for the conversion of RNA to

397

cDNA and a separate second reaction for qPCR amplification of the gene of

398

interest. The set of thermal profiles for two-step qRT-PCR is given as following.

399

Profile 1, directs cDNA synthesis by reverse transcriptase using a 30-minute,

400

42°C synthesis protocol. Profile 2, Segment 1 directs a 95°C incubation, used for

401

qPCR enzyme activation, and Segment 2 directs 40 cycles of qPCR amplification.

402

To quantify the expression level of the genes of interest, the Ct values of

403

both the sample and GAPDH were calculated by setting a cutoff of the relative

404

fluorescence intensity. The threshold value has to be set at a point where all

405

samples being analyzed display the same rate of increase in the fluorescence

406

intensity, and ideally this increase responds to an exponential function. We used

19



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407

the amplification-based threshold algorithm (Default Method in the MxPro QPCR

408

Software) to determine the Ct value automatically. Then the change of RNA

409

expression level is calculated as follows:

410

CtSample-CtGAPDH=∆Ct

411

Relative sample RNA expression level = 2-∆Ct

412

RNA expression level change = 2-∆Ct(experimental)/2-∆Ct(control)

413

Image acquisition and analysis

414

Briefly, all images were taken on a custom Olympus IX83 inverted

415

microscope equipped with a 100X UPlanSApo, N.A. = 1.49, oil-immersion phase

416

objective and Andor iXon Ultra897 EMCCD. The details about the configurations

417

of the custom microscope was listed in Supplementary Table 10. The microscope

418

stage incubation chamber was maintained at 37 °C and 5% CO2. A 405-nm laser

419

was used to excite BFP and DAPI; a 488-nm laser was used to excite sfGFP; a

420

561-nm laser was used to excite Cy3; a 647-nm laser was used to excite Cy5.

421

The laser power was modulated by an acousto-optic tunable filter (AOTF). Cells

422

were washed three times by PBS and imaged in phenol red-free DMEM (Thermo

423

Fisher, 21063029) for live cell imaging. Movies of short time scale chromatin loci

424

dynamics in living MDA-MB-231 cells were acquired at 50 ms exposure time (20

425

Hz) with only one layer. We used highly inclined thin illumination (HILO) to avoid

426

stray-light reflection and to reduce background from cell auto-fluorescence in the

427

live cell tracking experiment. For the experiment to count the number of loci in

428

each cell and the percentage of positive cell, cells were fixed by 4% PFA and

429

stained by DAPI for 2 min. Then images were acquired in PBS by 30 layers with a

20


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430

step size of 0.5 µm, each layer with a 500 ms exposure time in wide-field

431

illumination mode.

432

Analysis of MSD and velocity autocorrelation was carried out using custom

433

MATLAB scripts. The MSD analysis method has been described previously 6. To

434

quantify the influence of localization error on the accuracy of MSD analysis, we

435

compared the localization error with displacement of successive frame. The

436

localization error is calculated as followed: A , = B + × 2πσ σ

437

s = (σx + σy)/2

438

Where B is the background level, A is the height of the Gaussian peak, x0 is

439

the x center of the Gaussian peak, y0 is the y center of the Gaussian peak, σx is

440

the x standard deviation, σy is the y standard deviation, and s is the localization

441

error.

442 443

We then calculated all displacements between of two successive frames in the trajectories as followed: = ∆0 2 + ∆0 2 #$

444

where and ∆% and ∆% were the difference between two successive frames.

445

Average velocity autocorrelation function was calculated as described

446

previously by Joseph S. Lucas et al. 24. The function can be expressed as follows: & ' ( ) = < +, + ) ∙ +, > +, =

/, + 0 − /, 0

21



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447

Where /, is the two-dimensional coordinate at time point ,, δ is the time interval

448

over which the average velocity is calculated and ) is the lag time over which the

449

correlation in velocities is calculated.

22


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450

AUTHOR INFORMATION

451

Corresponding Author

452

*Phone#: 86-10-62744060

453

Author Contributions

454

S.S. and Y.S. conceived and designed the experiments. S.S. and YA.S.

455

performed the cloning experiments. S.S., L.C. and Y.H. performed the FISH

456

experiments. S.S. and X.F. performed the qPCR experiments. S.S. analyzed all

457

the data. S.S. and Y.S. wrote the manuscript.

458

Notes

459

The authors declare no competing financial interests.

460

ACKNOWLEDGMENTS

E-Mail: [email protected]

461

We thank Dr. Ronald D. Vale (University of California, San Francisco) for

462

providing SunTag plasmids, Dr. Wensheng Wei (School of life sciences, Peking

463

University) for providing sgRNA expression plasmids, Dr. Feng Zhang (Broad

464

Institute) for providing plasmid px330 (Addgene Plasmid #42230), Dr. Scot Wolfe

465

(University of Massachusetts Medical School) for pHAGE-EF1α-dCas9-KRAB

466

(Addgene Plasmid #50919), Dr. George Church (Harvard University) for SP-

467

dCas9-VPR (Addgene Plasmid #63798) and Dr. Wei Guo (Department of Biology,

468

University of Pennsylvania) for providing the MDA-MB-231 cell line. We also

469

thank Ms Xuefang Zhang from the National Center for Protein Sciences Beijing

470

(Peking University) for her excellent assistance with FACS. This work is

471

supported by grants from the 863 Program SS2015AA020406 and National

23



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472

Science Foundation of China 21573013, 21390412, 31271423 and 31327901, for

473

Y.S.

24


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474

SUPPORTING INFORMATION

475

Supplementary protocol 1, Supplementary Figures 1-12, Supplementary Tables

476

1-10, and Supplementary Plamid Maps 1-5.

477

REFERENCES

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Figure 1. Hierarchical assembly of multiplexed sgRNA expression plasmid. (A). Workflow for the hierarchical assembly of 20 sgRNAs. U6 promoter and sgRNA scaffold are PCR amplified and then ligated with each annealed oligo in 20 separated Golden Gate reactions with a type IIs restriction endonuclease BsmBI (Step 1). Each Golden Gate reaction mix was then used as template for amplification without purification by 20 pair of primers, which can be grouped into 4 categories, each member in a group with different adaptors (Step 2). The purified 20 sgRNAs expression cassettes were then separated into 4 group and implemented with the second round Golden Gate reaction using BsaI (Step 3). The four fragments each carried 5 sgRNAs expression cassettes were purified and then ligated together into a third round Golden Gate reaction with a receptor plasmid using BpiI (Step 4). Due to the repetitive sequence of each sgRNA expression cassette, each step should be verified by agar gel electrophoresis to guarantee that each reaction was carried out successfully. (B). A typical gel of the first round PCR result for the successful ligation of U6, annealed oligos, sgRNA scaffold. Trans2K plus II DNA ladder (TransGen Biotech) was shown in the left. The 20 individual sgRNA



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expression cassettes all show the expected length on the gel, indicating that this cloning method is quite robust. A detail to note is that the first and fifth sgRNA in each group are slightly longer than the remaining one. This is because two handles are added to the 5’ of the first sgRNA and 3’ of the fifth sgRNA to facilitate the PCR of the five sgRNAs in the following step. (C). A typical gel of the second round PCR result for the successful ligation of five sgRNA expression cassettes into one fragment. Trans2K plus II DNA ladder was shown in the left. Due to the repetitive nature of the five sgRNAs fragment, laddering effect was observed. The indicated repeat number was shown in the right. (D). A typical gel of the verification of the final all-in-one plasmid. Trans15K DNA ladder (TransGen Biotech) was shown in the left. All the plasmids were linearized by endonuclease enzyme BsaI. The empty vector was also shown as a control. All the tested six plasmids have the right insertion size, suggesting the high efficiency of the assembly process.

174x237mm (300 x 300 DPI)


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Figure 2. Single non-repetitive sequence labeling by multiplexed sgRNAs expression. (A). Schematic diagram of a non-repetitive sequence labeling of the genome. With the cell line that expressed proper level of SunTag system and 20 different sgRNAs tiling a short range sequence, arbitrary target loci can be labeled. The all-in-one plasmid carries 20 sgRNAs expression cassette each with a U6 promoter and poly U terminator so that individual sgRNA can be expressed independently. Six sgRNAs are shown as a demo here. The background fluorescent signal arises from the unbound dCas9 with multiple sfGFP molecules and the free sfGFP single molecules. Minimal level and proper stoichiometry (1:24) of SunTag system is the key point for the successful labeling. In principle, the 20 sgRNAs can direct 20 dCas9sunTag molecule, that is 480 sfGFP molecules, to the target loci to gather the fluorescent signal. (B). Workflow for the establishment of live imaging system with proper expression level. Piggybac plasmids dCas9-(GCN4)X24-P2A-BFP and scFV-sfGFP were used to construct the stable cell line. The MDA-MB-231 cells were co-transfected with SunTag expression plasmids in PiggyBac backbone and transposase expression plasmid, followed by puromycin and hygromycin selection for about 2 weeks. sfGFP and BFP



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double positive single clones with minimal background expression and high labeling efficiency were selected and transient transfected with multiple sgRNA expression plasmid to test the performance of each clone in subsequent live imaging experiment. (C). The labeling result of non-repetitive sequence in human cells. By transfection of 20 sgRNAs from an allin-one plasmid targeting the first intron of human MUC4 gene, bright fluorescent puncta (indicated by arrow) can be visualized. A Cy5-labeled probe was used to perform DNA fluorescence in situ hybridization against the third exon of MUC4 to verify the labeling result of CRISPR. The nucleus was stained with DAPI. The merged image showed the colocalization of CRISPR signal and FISH signal. The region targeted by CRISPR is about 1.5 kb which has 20 binding sites, while the region targeted by FISH is about 9 kb which has 67 binding sites. Because the FISH probes target region is a repetitive sequence, so we only need one probe to get high signal-to-noise ratio image. All scale bars are 5 µm. (D). Single non-repetitive sequence can be labeled in different cell cycle stages. In G1 of interphase, two loci can be visualized. In G2 of interphase, four loci appeared as closely located pairs. During mitosis, the duplicated loci were allocated into two cells. In anaphase and telophase, the position of MUC4 loci nearly mirrored each other. All scale bars are 10 µm.

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Figure 3 Live cell tracking of single non-repetitive genomic locus. (A). Typical image and trajectories of the MUC4 locus in live cell. Trajectories were displayed by the colorcoded time points with green for the starting, blue for the intermediate and red for the ending frames. The time lookup table was in the right. Scale bar: 5 µm. (B). Time-averaged MSD plotted as a function of time lag (delay) for the MUC4 region in live cells. MSD was shown for delay up to 10 % of total imaging time. 17 trajectories were analyzed individually (left) or averaged among all the trajectories (right). Dashed lines indicate a subdiffusive scaling exponent (α) of 0.34, demonstrating the locus was undergoing constraint diffusion. (C). Velocity autocorrelation analysis of MUC4 locus in live cells. Velocity was calculated over discretization intervals (δ) ranging from 0.05 s to 5 s in 0.05 s intervals (left). Velocity autocorrelation curves for different values of δ, plotted against a rescaled timelag (t/δ) (right). The color scheme represents the values of δ from small (blue) to large (red).

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Figure 4 Synergy activation of the endogenous genes by multiplexed sgRNAs targeting one region. (A). Synergy gene activation by 20 sgRNAs targeting the promoter region of SOX2 gene. HEK293T cells were transfected with either individual plasmid (single or all-in-one) or cocktail of five plasmids. dCas9SunTag-VP64 are used for the maximum activation efficiency. qRT-PCR were used for the quantification of the activation results. (B). Synergy gene repression by 20 sgRNAs targeting the promoter region of SOX2 gene in HEK293T cells. The same plasmids are used as in (A) except dCas9-KRAB are used instead. Cells without transfection were used as control. All values are mean ± s.e.m. with n = 3 biological replicates.

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Figure 5 Multiplexed modified sgRNA expression activate and repress multiple endogenous genes simultaneously. (A). 10 sgRNA-MS2 (2.02) and 10 sgRNA-PP7 (2.02) were assembled into one plasmid. dCas9, tdPCP-VPR, tdMCP-KRAB and all-in-one sgRNA expression plasmid were cotransfected to HEK293T cells. 10 genes were activated and 10 other genes were repressed simultaneously in one cell. (B). HEK293T Cells were analyzed by qRT-PCR 2 days after transfection. The genes that are targeted with sgRNA-PP7 (2.02) were upregulated while the gene that are targeted with 10 sgRNA-MS2 (2.02) were downregulated without crosstalk. All values are mean ± s.e.m. with n = 3 biological replicates.

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Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive

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