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Rational design of mini-Cas9 for transcriptional activation. Dacheng Ma1, Shuguang Peng1, Weiren Huang2, Zhiming Cai2 and Zhen Xie1*. 1MOE Key ...
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Letter Cite This: ACS Synth. Biol. 2018, 7, 978−985

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Rational Design of Mini-Cas9 for Transcriptional Activation Dacheng Ma,† Shuguang Peng,† Weiren Huang,‡ Zhiming Cai,‡ and Zhen Xie*,† †

MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and System Biology, Department of Automation, Tsinghua National Lab for Information Science and Technology, Tsinghua University, Beijing 100084, China ‡ State Engineering Laboratory of Medical Key Technologies Application of Synthetic Biology, Shenzhen Second People’s Hospital, the First Affiliated Hospital of Shenzhen University, Shenzhen, China

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S Supporting Information *

ABSTRACT: Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications. KEYWORDS: CRISPR/Cas9, gene activation, AAV, VPR glutamine tRNA can be used to replace the ∼250-bp RNA polymerase III promoter to drive expression of the tRNA:gRNA fusion transcript that is cleaved by endogenous tRNase Z to produce the active gRNA.20 These efforts facilitate the construction of an all-in-one AAV delivery vector for in vivo applications of the CRISPR/Cas technology.15,17,21 Recent structural studies of SpCas9, SaCas9, and AsCpf1 have elucidated functions of conserved domains among these class 2 CRISPR effectors, including a recognition (REC) domain, a protospacer adjacent motif (PAM) interacting (PI) domain, and nuclease domains.22−25 The HNH/NUC and RuvC nuclease domains, respectively, cleave the complementary and the noncomplementary DNA strands.22−25 Interestingly, the truncated SpCas9 mutant without HNH domain displays nearly intact DNA binding activity, while the truncated SpCas9 mutant without the REC2 domain retains half of the wild-type cleavage activity.23,24 To improve genome editing specificity, the FokI nuclease domain is fused to the N-terminal of the dCas9 in the PAM-out orientation, which dimerizes with the guidance of two gRNAs and causes DNA cleavage.26 However, the length of these dCas9 fusion genes usually impede direct loading into a single AAV vector due to the restrictive cargo size.

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he CRISPR/Cas9 system enables programmable and precise genetic manipulations, providing viable opportunities for personalized therapies.1−4 Through mutation of the active residues related to the nuclease function, the Cas9 protein can be engineered into the nuclease-null or “dead” Cas9 variant (dCas9) that only retains the DNA binding ability but lacks detectable nuclease activity.5 dCas9 can be fused to different domains for efficient transactivation, such as the four tandem copies of herpes simplex viral protein 16 (VP64), the repeating SunTag peptide array and the tripartite activator VP64-p65-Rta (VPR).6−8 The dCas9-based transactivator for specific gene activation has been used to activate the expression of endogeneous gene for ameliorating disease phenotypes and induce cell differentiation.9,10 Although the AAV vector has a limited payload capacity, which is inefficient above 5-kb, it is still an attractive delivery vehicle for the CRISPR/Cas9 system because of low pathogenic risk, reduced immunogenicity, and a wide range of tissue tropism.11−13 To bypass the AAV payload limit, the 4.2-kb Cas9 from Streptococcus pyogenes (SpCas9) is split and packaged into two separate AAVs along with the guide RNA (gRNA) expression unit, which allows functional reconstitution of full-length SpCas9 in vivo.13,14 Nevertheless, this dual-AAV system may reduce the delivery efficiency. Another strategy is to search for natural class 2 CRISPR effectors with a diminished size, such as the 3.2-kb SaCas9, ∼3-kb CasX, and CjCas9 identified in uncultivated organisms by using metagenomic data sets.15−19 To further reduce the transgene size, the ∼70-bp © 2018 American Chemical Society

Received: November 12, 2017 Published: March 21, 2018 978

DOI: 10.1021/acssynbio.7b00404 ACS Synth. Biol. 2018, 7, 978−985

Letter

ACS Synthetic Biology

Figure 1. Rational Design of the Compact CRISPR/Cas9 System. (A) Diagram of EBFP2 transcription activation assay for the compact Cas9 derivatives fused with the VPR domain. The constitutively expressed mKate2 was used as a transfection control. (B) Diagram of dSpCas9, minidSpCas9−1 and mini-dSpCas9−2 domain organization (left) and their corresponding gene activation efficiency (right). (C) The schematic of domain organization of dSaCas9 and SaCas9 derivatives are shown on the left, and the results of their gene activation efficiency are shown on the right. (D) Diagram of dAsCpf1 and mini-AsCpf1−1 domain organization (left), and the corresponding gene activation efficiency (right). (B−D) Data are shown as the mean ± SEM fold change of EBFP2 fluorescence from three independent replicates measured by using flow cytometer 48 h after transfection into HEK293 cells.

optimized guide RNA-2 was ∼8-fold higher than the result when the wild type guide RNA was used by introducing both of the two point mutations in the putative RNA Pol III terminator sequences (SI Figure 1A). On the basis of this mutant SaCas9 being retargeted by optimized guide RNA, we constructed the mini-SaCas9−1:VPR by replacing the conserved REC-C domain (Δ234−444) with a “GGGGSGGGG” linker (GSlinker), which only retained ∼21% transactivation activity of the dSaCas9:VPR (Figure 1C and SI Figure 2B). Although the GS-linker is widely used as a flexible linker, it may still distort the SaCas9 structure. Inspired by a recent computational protocol called SEWING,31 we developed an adjacent residue searching (ARS) protocol to search for existing structures between discontinued Cas9 fragments (details in the methods section). Replacement of the GS-linker with a “KRRRRHR” (R-linker) from the SaCas9 BH domain that appropriately filled in the REC-C deletion gap by using the ARS protocol, resulted in a 4-fold increase in the transactivation capacity of mini-SaCas9−2 (Figure 1C and SI Figure 2B). Interestingly, the mini-SaCas9−3 that was generated by deleting the REC-C domain without any linker displayed a similar transactivation efficiency to the mini-SaCas9−2 (Figure 1C and SI Figure 2B). We also found that a “GSK” linker, derived from a putative gene (accession number, O67859) in Aquifex aeolicus by using the SAR protocol, fit the deletion gap of the HNH domain (Δ479−649). The resulted mini-SaCas9− 4 exerted a stronger transactivation efficiency than the dSaCas9:VPR (Figure 1C and SI Figure 2B). However, the

In this study, we constructed a set of downsized variants of SaCas9 by deleting conserved functional domains, which retained the DNA binding activity but displayed no DNA cleavage activity. We also constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which allow us to produce a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette.

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RESULTS AND DISCUSSION

We first constructed two mini-dSpCas9 genes by respectively deleting the C-terminal region of REC1 domain (REC-C, Δ501−710) and the HNH domain (Δ777−891) that may be dispensable for DNA binding activity of the dCas9, and respectively fused to the VPR transactivation domain. By using our previously established reporting system in cultured human embryonic kidney 293 (HEK293) cells (Figure 1A),27 we demonstrated that the two mini-dSpCas9:VPR variants retained more than 50% of transactivation capacity compared to the dSpCas9:VPR (Figure 1B and SI (SI) Figure 2A). Recently, the wildtype SaCas9 has been engineered to recognize an altered PAM (“NNNRRT”).28 It has been shown that introducing an A-U flip or a U-G conversion to disrupt the putative RNA Pol III terminator sequences in the first stem loop of the gRNA scaffold enhances the efficiency of the dCas9-mediated DNA labeling.29,30 We demonstrated that the endogenous transactivation efficiency of dSaCas9:VPR for the IL1RN gene with 979

DOI: 10.1021/acssynbio.7b00404 ACS Synth. Biol. 2018, 7, 978−985

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ACS Synthetic Biology

Figure 2. Effect of the compact SaCas9 derivatives on DNA cleavage. (A) Diagram of the EYFP reconstitution assay to evaluate the DNA cleavage efficiency. (B) Domain organization of dSaCas9 and mini-dSaCas9−4 fused with the FokI domain at the N terminal are shown in the upper panel. Domain organization of split dSaCas9 or split mini-dSaCas9−4 fused with the FokI domain in the middle is shown in the lower panel. (C) DNA cleavage efficiency by the FokI:dSaCas9, the FokI:mini-dSaCas9−4, and the split fusions of FokI and dSaCas9 with or without the HNH domain, with a spacer length ranging from 12-bp to 24-bp. Each bar shows mean fold changes (mean ± SEM; n = 3) of EYFP fluorescence measured by using flow cytometer 48 h after transfection in HEK293 cells. (D) The DNA cleavage assay targeting the human CCR5 gene by SaCas9, SaCas9-Nick, FokI:dSaCas9, FokI:mini-dSaCas9−4, and the split fusions of FokI and dSaCas9 with or without the HNH domain.

promoter by coexpressing the optimized gRNA. The result showed that the mRNA level was increased at least 1000-fold through coexpressing the optimized guide RNA-2 and dSaCas9:VPR (SI Figure 4). We demonstrated that all compact SaCas9:VPR variants coexpressed with the optimized guide RNA-2 can activate the IL1RN gene expression. The miniSaCas9−4:VPR generated a comparable IL1RN gene activation level to that of the dSaCas9:VPR (SI Figure 4). As shown in Figure 2A, we used a similar EYFP reconstitution assay to evaluate DNA cleavage efficiency of dSaCas9 derivatives fused with FokI along with two truncated gRNAs that respectively contain 18-nt sequences complementary to the target.27 We demonstrated that the FokI:minidSaCas9−4 that was made by fusing the FokI domain to the Nterminal of the mini-dSaCas9−4 displayed a similar DNA cleavage activity compared to the FokI:dSaCas9 control, with a spacer between two gRNA complementary regions ranging from 12-bp to 24-bp in the PAM-out orientation (Figure 2B− C). We then searched for another appropriate FokI insertion position in the mini-dSaCas9−4. However, the predicted distance between the N-terminal and the C-terminal of the FokI nuclease domain was 35 Å (SI Figure 5), which makes it challenging to find an appropriate insertion position in the middle of the dSaCas9. Alternatively, we split mini-dSaCas9−4 at residue 733 and fused the FokI after the splitting point with a triplicate G-linker. We further removed the first four residues of

2-kb mini-SaCas9−5 by deleting both the REC-C domain and the HNH domain only retained 41% transactivation activity over the wild-type control (Figure 1C and SI Figure 2B). Interestingly, the transactivation efficiency of miniSaCas9−5 was increased ∼30-fold by optimized guide RNA than wild type guide RNA (SI Figure 3A). In addition, we observed ∼50% transactivation activity when we used glutamine tRNA instead of the U6 promoter to drive gRNA expression (SI Figure 3B). To evaluate the DNA cleavage efficiency of mini-SaCas9 variants, we used a reporter reconstitution assay as described in our previous study,27 in which DNA cleavage can trigger the reconstitution of the active enhanced yellow fluorescent protein (EYFP) reporter gene from the inactive form (SI Figure 2C). We demonstrated that either deleting the REC-C or deleting both the REC-C and HNH domains resulted in a background EYFP expression, suggesting that the domain deletion abolished the DNA cleavage activity of the mini-SaCas9 variants (SI Figure 2D). In addition, we showed that deleting the REC2 domain (Δ324−525) retained 46% transactivation capacity of AsCpf1:VPR but abolished the DNA cleavage activity, suggesting that this deletion strategy is applicable for distinct class 2 CRISPR effectors (Figure 1D and SI Figure 2E− G). To assay whether the compact SaCas9 variants can efficiently activate expression of the endogenous gene, we retargeted the miniCas9:VPR variants and dSaCas9:VPR to the IL1RN 980

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Figure 3. Construction of the compact VTR and MoonTag Systems for transcription activation. (A) Schematic representation of VPR, VTR1, VTR2, VTR3, and VP64 transcription activation domain and their corresponding gene activation efficiency evaluated by using the EBFP2 reporting system shown in Figure 1A. Unpaired t-test was performed for comparison between indicated samples. The asterisks (∗∗∗∗) indicate p < 0.0001. (B) Schematic representation of the SpyTag and MoonTag repeating array for transcription activation. The corresponding gene activation efficiency was evaluated by using the EBFP2 reporting system shown in Figure 1A. (A and B) The optimized gRNA-2 was used in the experiments. Data are shown as the mean fold change (mean ± SEM; n = 3) of EBFP2 fluorescence measured by using flow cytometer 48 h after transfection into HEK293 cells.

partner, called SpyCatcher (SI Figure 6C).34 To construct a repeating peptide array with a smaller size than the SunTag system,8 we fused four tandem repeats of SpyTag to the Cterminal of mini-SaCas9−5 and fused the SpyCatcher with the VPR domain, allowing spontaneous assembly of a VPR transactivation scaffold in cells (Figure 3B). We showed that the SpyTag system induced the expression of the enhanced blue fluorescent protein 2 (EBFP2) reporter gene to 100-fold compared to the negative control (Figure 3B). To search for the homologue of SpyTag and SpyCatcher, we found that a putative protein (accession no. WP_054278706) from Streptococcus phocae shared 60% sequence similarity to the FbaB (SI Figure 6C). We hypothesized that this protein can be split similarly as the SpyTag and SpyCatcher. We developed a similar scaffold system called the MoonTag system by fusing four tandem repeats of the 13-residue MoonTag to the miniSaCas9−5 and making a hybrid of the MoonCatcher and VPR domains (Figure 3B). Although the MoonTag system was not orthogonal to the SpyTag system, the MoonTag system was 5fold more efficient to activate the EBFP2 expression (Figure 3B). The AAV load size limitation can be overcome by splitting the Cas9 into an N terminal and a C terminal fragment separately expressed by two single AAV vectors. We previously demonstrated that intein-mediated split Cas9 reconstitution displayed enhanced activity compared with their counterparts without intein fusion.27 Next, we sought to compare the efficiency of the previous double-AAV-vector system by coexpressing split Cas9 fragments with the all-in-one AAV system using the compact Cas9 variants. First, we split the dSaCas9 at residue 739, fused the N and C fragments with intein-N and intein-C respectively. The dSaCas9N:Intein-N driven by the constitutive CMV promoter and the U6-driven original gRNA targeting the TRE promoter were loaded into one AAV vector (AAV-splitN), while the CMV-driven dSaCas9C:VPR was loaded into the second AAV vector (AAV-splitC) (Figure 4A). In the all-in-one AAV system, a single AAV virus (AAV-single-1) encoded a constitutively expressed mini-SaCas9−5:VTR1 and an optimized gRNA

the C-terminal fragment (Δ734−737) that might interfere with the reconstitution of two split fragments. The split dSaCas9 or split mini-dSaCas9−4 without the HNH domain resulted in ∼3% to 33% of the EYFP expression level induced by the wildtype SaCas9, less than ∼20% to 62% induce efficiency of FokI:dSaCas9 or FokI:mini-dSaCas9−4, with a spacer ranging from 12-bp to 24-bp in the PAM-out orientation (Figure 2B,C). In addition, we evaluate the DNA cleavage activity of these FokI fusions when targeting the human CCR5 gene in HEK293 cells by using the T7 Endonuclease I (T7E1) assay.32 The results showed that all fusions of FokI and dSaCas9 with or without the HNH domain triggered the DNA cleavage on the CCR5 gene, although deleting the HNH domain reduced DNA cleavage efficiency (Figure 2D). Next, we sought to engineer compact transcription activators based on dCas9-VPR.7 The entire P65 contains a DNA binding domain in the N-terminal, and two transactivation domains (TA1 and TA2) in the C-terminal.33 However, only the TA2 and the partial TA1 are included in the tripartite VPR domain.7 To reduce the size of the dCas9-VPR, we constructed the miniSaCas9−5:VTR1 by replacing the P65 domain in the VPR with the TA1 and TA2 domains (Figure 3A). We further substituted the P65 domain in the VPR with two repeats of the TA1 domain, termed VTR2 (Figure 3A). We demonstrated that the VTR1 and VTR2 domains retain 47% and 55% transactivation efficiency of the VPR domain (Figure 3A). We further removed the partial RTA domain, resulting in a 0.8-kb transactivation domain (VTR3), which retained 53% transactivation efficiency of the VPR domain (Figure 3A). We also found the miniSaCas9−5:VTR3 paired with the optimized gRNA showed 80% transactivation efficiency compared to the split SaCas9:VPR paired with original gRNA (SI Figure 6A,B). In addition, we compared the transactivation efficiency of the VTRs with the VP64 domain fused to the mini-SaCas9−5 only. The result showed that fusion of the different transactivation domain behind the VP64 domain enhanced the activation efficiency (Figure 3A). Recently, a 13-residue SpyTag derived from FbaB has been shown to form a covalent bond with its 116-residue binding 981

DOI: 10.1021/acssynbio.7b00404 ACS Synth. Biol. 2018, 7, 978−985

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Figure 4. Construction of the compact CRISPR/Cas system for transcription activation delivered by AAV vector. (A) Schematic representation of the all-in-one AAV system and the double AAV system. AAV-single-1 contains the mini-SaCas9−5:VTR1 and U6-driven optimized gRNA-2, and the split double AAV system consists of AAV-splitN and AAV-splitC. AAV-splitC encodes the inteinC, dSaCas9 C-terminal fragment and VPR domain driven by the CMV promoter. AAV-splitN encodes the dSaCas9 N-fragment and inteinN driven by the CMV and the U6-driven gRNA. (B) EBFP2 activation ratio in HEK293 cells infected by either AAV-single-1 or the split double AAV system. Each bar shows the mean ± SEM (n = 3) of % EBFP2+ measured by using a flow cytometer 4 days after infection. The %EBFP2+ ratio was calculated by using the total number of EBFP2 positive cells divided by the frequency of the mKate2 positive cells (Q2 + Q3). The plasmid DNAs that encode the EBFP2 reporter gene and the mKate2 control gene were introduced into HEK293 cells by transient transfection, 1 day before the AAV infection. An unpaired t-test was performed for comparison between indicated samples. The asterisks (∗∗) indicate p < 0.01. (C) Representative scatter plots of FACS results in panel B.

DNA cleavage activity by deleting conserved HNH and/or REC-C domains based on the structural information (Figure 1). Recently the single molecular studies showed that both of the HNH nuclease domain the REC-C terminal domain control the conformation of the RUC nuclease domain activity which may be the possible reason why these set of compact Cas9 derivatives have no DNA cleavage activity.23,35 In addition, we provided a novel strategy to engineer the dimeric gRNA-guided nuclease by splitting the mini-dSaCas9 and fusing the FokI domain right after the split point (Figure 2). We also observed that the DNA cleavage efficiency of each FokI fusion Cas9 is different when the spacer distance changes and when targeting endogenous gene (Figure 2). Further experiments are needed to quantify this difference and optimize the DNA cleavage efficiency in different situations. The VPR domain was made by fusing p65, Rta, and VP64. However, Rta and p65 contain DNA binding and transcription activation domains that can initiate promiscuous genome interactions and gene activations.33,36 We developed a series of compact transactivation domains (VTR1, VTR2, and VTR3) by deleting the DNA binding domains in Rta and p65 (Figure

recognizing the TRE promoter (Figure 4A). We introduced a TRE-driven EBFP2 reporter gene into HEK293 cells by transient transfection (Figure 4A), followed by infection of the indicated AAV viruses. An AAV virus containing the miniSaCas9−5:VTR1 and optimized gRNA-2 recognizing off-target site instead of the TRE promoter was added as the negative control. After 4 days, we observed ∼40% transfected cells were activated by AAV-single-1. Different stoichiometric ratios of the double-AAV system were tested, and the 70:30 ratio yielded a maximum cell activation of ∼30% (Figure 4B,C). However, the overall EBFP2 level induced by AAV-single-1 was about 3 fold less than the that resulted by using AAV-splitN and AAV-splitC with a 90:10 ratio (SI Figure 7A), which was likely because the transactivation of the VTR1 domain was less than the VPR domain (Figure 3). We also made three all-in-one AAV variants (AAV-single-2, -3, and -4) by replacing the VTR1 domain with either VTR2 or VTR3, using either the CMV or EFS promoter (SI Figure 7B,C). However, the ratio of the activated cell decreased, suggesting that AAV-single-1 is more suitable for transactivation with a high infection efficiency. In this study, we engineered a set of compact Cas9 derivatives that retained efficient DNA binding activity but no 982

DOI: 10.1021/acssynbio.7b00404 ACS Synth. Biol. 2018, 7, 978−985

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To extract the viral genome, 1 μL of purified AAV virus was incubated with 1 U of DNase I (NEB) in a 50-μL reaction containing 25 mM of Tris-HCl (pH 7.4) and 10 mM of MgCl2 at 37 °C for 30 min, and then incubated at 75 °C for 10 min. Then the reaction was treated with 200 μg of proteinase K in the presence of 5 mM Tris-HCl (pH = 8.0), 10 mM Na2EDTA, and 20 mM of NaCl2 at 37 °C for 1 h. A 10-fold serial dilution of the corresponding plasmid DNA used for AAV virus package was made between 0.2 ng/μL and 0.02 pg/μL for virus titer measurement. The AAV titer was determined by quantitative PCR using 2× EvaGreen Master Mix (Syngentech). HEK293 cells were seeded in 24-well plates at a density of 1× 105 cells/ well. After 1 day, cells were transfected with the plasmid DNAs that encode the EBFP2 reporter gene, and the mKate2 gene that served as an internal control (SI Table 3). Then, purified AAV was added to each well 1 day after transfection. Cells were cultured for additional 4 days before flow cytometry and imaging analysis. Flow cytometry. Cells were trypsinized 48 h after transfection and centrifuged at 300g for 7 min at 4 °C. The supernatant was removed, and the cells were resuspended in 1× PBS that did not contain calcium or magnesium. Fortessa flow analyzer (BD Biosciences) was used for fluorescence-activated cell sorting (FACS) analysis with the following settings. EBFP2 was measured using a 405 nm laser and a 450/50 filter with a photomultiplier tube (PMT) set at 275 V. The EYFP was measured with a 488 nm laser and a 530/30 filter using a PMT set at 270 V. The mKate2 was measured with a 561 nm laser and a 670/30 filter using a PMT set at 350 V. The iRFP was measured with a 640 nm laser and a 780/60 filter using a PMT set at 480 V. For each sample, ∼1 × 104 to ∼5 × 104 cell events were collected. The relative fluorescence intensity of EBFP2 is defined as the average fluorescence intensity of EBFP2 divided by the average fluorescence intensity of the internal control fluorescence (such as mKate2). “Fold change (EBFP2)” was defined as the average of relative fluorescence intensity of EBFP2 of samples divided by the that of the control samples. “%EBFP2+” is calculated as the fraction of positive EBFP2 cell divided by the fraction of positive mKate2 cell to normalize the transfection difference. Fluorescence Microscopy. Approximately 48 h after transfection, fluorescent images of cultured HEK293 cells were captured by using Leica DMi8 microscope with the 10× objective lens. The EBFP2 fluorescence was observed by using a DAPI filter cube with excitation at 350/50 nm, dichroic at 400 nm and emission at 460/50 nm. The mKate2 fluorescence was observed with the TXR filter cube with excitation at 560/40 nm, dichroic at 585 nm, and emission at 630/75 nm. Image acquisition and postacquisition analysis were performed using the LASX software suite (Leica). Adjacent Residue Searching. The BLASTP program was used to search for suitable linker sequences in the NCBI PDB database to close the domain deletion gap between the Nterminal and C-terminal anchors.37 The query sequences were composed of the last three amino acid residues upstream of the N-terminal anchor and the degenerate residue sequences with an estimated length and the first three amino acid residues downstream of the C-terminal anchor. The candidate linker sequences were selected if the following three criteria were satisfied: (1) its N-terminal region shared high structural similarity with the N-terminal anchor region based on the structure similarity; (2) its C-terminal region shared high structural similarity with the C-terminal anchor region based on

3), which may benefit for a more specific transactivation than the VPR domain. Our study highlighted a practical approach to load all essential elements of the CRISPR/Cas transactivation system in one AAV system (Figure 4). Because of the AAV cargo restriction, only one gRNA was used in the all-in-one system. For endogenous gene activation, multiple gRNAs targeting the same promoter region can induce higher gene expression. New strategies are needed for expressing multiple gRNAs in the allin-one system, such as the tRNA driven and self-splicing system. In summary, our downsized CRISPR/Cas9 and gene activation systems will be particularly appealing in biomedical applications that require safe and efficient delivery in vivo.

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MATERIALS AND METHODS Reagents and Enzymes. Restriction endonuclease, polynucleotide kinase (PNK), T4 DNA ligase, Quick DNA ligase, and Q5 High-Fidelity DNA Polymerase were purchased from New England Biolabs. Oligonucleotides were synthesized by Genewiz and Sangon Biotech. Plasmid DNA Constructs. The gRNA sequences were listed in SI Table 1. The protein sequences of Cas9 derivatives and domains used in this study were summarized in SI Table 2. Cell Culture and Transfection. The HEK293 cell line was purchased from Life Technologies. HEK293 cells were cultured in high-glucose DMEM complete media (Dulbecco’s modified Eagle’s medium (DMEM), 4.5 g/L glucose, 0.045 unit/mL of penicillin, 0.045 g/mL streptomycin, and 10% FBS (Life Technologies)) at 37 °C, 100% humidity, and 5% CO2. One day before transfection, ∼1.2 × 105 HEK293 cells in 0.5 mL of high-glucose DMEM complete media were seeded into each well of 24-well plastic plates (Falcon). Shortly before transfection, the medium was replaced with fresh DMEM complete media. The transfection experiments were performed by using Attractene transfection reagent (Qiagen) by following the manufacturer’s protocol. The amount of plasmid DNAs used in transfection experiments is listed in SI Table 3. Cells were cultured for 2 days before flow cytometry and imaging analysis. AAV Packaging, Purification, and Infection. For AAV production, ∼1.2 × 106 HEK293 cells were seeded into 10 cm Petri dish. The pAAV-DJ serotype packaging DNA construct, pHelper construct, and pAAV construct were purchased from Cell Biolabs. Cells were cotransfected with 3 μg of pAAV-DJ, 3 μg of pHelper, and 3 μg of pAAV plasmid DNA carrying the gene of interest by using Attractene transfection reagent (Qiagen). After 3 days, all media including trypsinized cells were harvested in a 50 mL sterile tube. About 0.1 volume of chloroform was added into the sterile tube, and the tube was shaken at 250 rpm/min at 37 °C for 1 h. Then NaCl was added to the mixture to make the final concentration at 1 M. After centrifugation at 12000 rpm/min for 15 min, the upper aqueous phase was transferred to a new 50 mL sterile tube and mixed with PEG-8000 (10% final w/v) followed by incubation on ice for 1 h. The mixture was centrifuged at 11000 rpm/min for 15 min. The pellet was resuspended in 1.2 mL of D-PBS (with Ca2+ and Mg2+) purchased from Beyotime. DNase and RNase (Solarbio) were added to the final concentration of 1 μg/mL, respectively, and the mixture was incubated for 30 min at room temperature. Equal volume of chloroform was added into the mixture, followed by centrifugation at 12000 rpm/min for 5 min. The upper aqueous phase was collected for the following AAV infection experiment. 983

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the structure similarity; (3) superposition of the candidate linker sequences into the deletion gap does not create any steric clashes. RNA Purification and Quantitative PCR. Total RNA from HEK293 cells was extracted with Trizol reagent (Life Tech) A 500 ng sample of RNA was reversed transcripted by ReverTra Ace qPCR RT Master Mix with gDNA Remover Kit (TOYOBO), and 1 μL of cDNA was used for each qPCR reaction, using the 2× EvaGreen Master Mix (Syngentech). qPCR primers were listed in SI Table 1. qRT-PCR was run and analyzed in the Light cycler 480 II (Roche), with all target gene expression levels normalized to β-actin mRNA levels. T7E1 DNA Cleavage Assay. DNA was harvested 72 h post-transfection using a genomic DNA extraction kit (TianGen) according to the manufacturer’s protocol. PCR was performed to amplify the fragment of the CCR5 gene by using two primers (5′-CGTGTCACAAGCCCACAGATATTT-3′ and 5′-GCACAGGGTGGAACAAGATGG-3′). All reactions were performed with the high-fidelity DNA polymerase (TsingKe) with the resulting products purified by using the Gel Extraction Kit (GenStar). T7E1 digestion was then performed in NEB Buffer 2 according to the manufacturer’s instructions. The image of the electrophoresis of the cleavage products was analyzed by using ImageJ software.

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ASSOCIATED CONTENT

* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00404. Additional figures and tables as described in the text (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zhen Xie: 0000-0001-8798-9592 Author Contributions

Z.X. and D.M. conceived of the ideas implemented in this work. D.M. performed experiments. Z.X., D.M., S.P., W.H., and Z.C. analyzed the data. Z.X. supervised the project. Z.X., D.M., and S.P. wrote the paper. Notes

The authors declare the following competing financial interest(s): Z.X., D.M. and S.P. have filed a patent application to State Intellectual Property Office of China based on the findings in this work.

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ACKNOWLEDGMENTS

The research is supported by the National Key Basic Research Program of China (2014CB745200), National Natural Science Foundation of China (31771483, 81772737), Shenzhen Municipal Government of China (JCYJ20170413161749433), and Basic Research Program of Tsinghua National Lab for Information Science and Technology. We thank members of Xie Lab for helpful discussions. We thank Fei Sun and Ting Zhu for insightful discussion. We thank Yuxi Ke for proof reading of our manuscript. 984

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DOI: 10.1021/acssynbio.7b00404 ACS Synth. Biol. 2018, 7, 978−985