Cas9 Assay for Site-Specific ... - ACS Publications

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Article

Label-free CRISPR/Cas9 Assay Enable Site-Specific Nucleic Acid Detection Jianyu Hu, Min Jiang, Rui Liu, and Yi Lv Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Label-free CRISPR/Cas9 Assay Enable Site-Specific Nucleic Acid Detection

Jianyu Hu, †, ‡ Min Jiang‡, Rui Liu‡ * and Yi Lv§ *

† College of Architecture & Environment, Sichuan University, Chengdu 610064, China. ‡ Key Laboratory of Green Chemistry & Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, China §Analytical & Testing Center, Sichuan University, Chengdu 610064, China. *Yi Lv, E-mail: [email protected]. Tel. and Fax: +86-28-8541-2798. Rui Liu, E-mail: [email protected]. Tel. and Fax: +86-28-8541-2398.

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

ABSTRACT Development of CRISPR/Cas9 system has become a revolutionary step for genome engineering by the virtue of modification of target genomes. However, many biological applications with CRISPR/Cas9 system are impeded by off-target effects and loci-dependent nuclease activity with various sgRNAs. Common-used label strategy-based CRISPR/Cas9 assays often suffer from possible disturbance to Cas9 activity and time-consuming label procedure. Herein, we for the first time propose a DNA-templated CuNPs based label-free CRISPR/Cas9 assay, with low LOD of 0.13 nM and rapid detection in 35 min after CRISPR/Cas9 cleavage. Besides, the site-specificity of DNA substrate was demonstrated. Through the proposed label-free strategy, single-base change at specific loci could lead to significant reduction of Cas9 cleavage effect, while the other common genetic modifications might be accepted by CRIPR/Cas9 system. Therefore, the proposed label-free Cas9 assay may provide a new paradigm for priori in-vitro CRISPR/Cas9 assay and exploration for the in-vivo biological application.

2


Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Clustered regularly interspaced short palindromic repeat (CRISPR) is an RNA-directed adaptive immune system existed in the bacterial and archaeal, which can induce site-specific double strand DNA (dsDNA) cleavage with an engineered single guide RNA (sgRNA).1-3 Among different CRISPR systems, CRISPR/Cas9 has been extensively used in genome engineering of mammalian cells, including genome editing4, drug delivery5, and genetic screen6. Recently, Li’s group has established multiple bioassays with CRISPR/Cas9 system, including mRNA sensing7 and single-nucleotide variation in mitochondrial DNA8. Site-specific break is the priority demand of all CRISPR/Cas9 applications, despite there is still remaining off-target effect leading to unexpected genome damage and unpredictable risk to health. 9-12 To enhance the fidelity of CRISPR/Cas9 system, Yin et al. found that portion of DNA replacement with sgRNA could alleviate the off-target effect, while Fu et al. found similar result carried out with truncated sgRNA.13,14 Whereas, the engineering sgRNA suitable for different genome is alterable, while the database of criteria is still insufficient to computational simulation.15,16 Moreover, it was proved that CRISPR/Cas9 system shown quite different efficiencies with different genomic loci.17 Therefore, there are urge demands for priori in-vitro Cas9 assay before practical applications, in order to site-specific detection and sgRNA sequence validation. An ideal priori in-vitro Cas9 assay need to satisfy two key criteria: (1) the sensitivity of proposed assay should be low enough to nanomolar level as Cas9 is single-turnover enzymes which is large excess needed to the substrate;18,19 (2) the time cost should be short enough for high-throughput analysis due to the large amount of pre-screen gene and sgRNA sequence. Traditional PCR and isothermal amplification reactions,20,21 next-generation sequencing methods22 suffer from time-consuming process. Recently, label strategies are the mainstream techniques in the CRISPR/Cas9 assay. 3



Page 4 of 26

Fluorescence resonance energy transfer (FRET) pair label strategy was initially utilized in the mechanism and kinetic research.23 But the FRET pairs label strategy cannot directly detect dsDNA break but only reflect the binding steps of Cas9 binding procedure with DNA substrate.24 Instead, radioisotope label-based gel electrophoresis has been regarded as standard method in Cas9 assay.10 Despite the robustness, the inconvenience of radioactive labelling procedure makes the operation more complicated and time cost more than 4h. In order to shorten the reaction time, Seamon et al. developed a versatile fluorophore/ quencher pair-based label strategy enable high-throughput analysis, although high concentrations of enzyme (200 nM) is still required.25 Liu et al. utilized Ru(bpy)32+ as electrochemiluminescent label to evaluate CRISPR/Cas9 function with 1 nM of limit of detection (LOD) for Cas9.26 Besides, Cox et al. built up a universal sensitive method for CRISPR-associated nucleases by fluorescent RNA/DFHBI complex.27 Despite the success, the above strategies all have to go through label procedure to generate sensing unit. The possible disturbance to Cas9 activity, extra operation procedure, and additional time-cost hinder the label strategies becoming ideal priori Cas9 assay methods. Label-free bioassays draw a lot of attentions in recent years, because of facile procedures, time-saving and direct interaction information deliver between sensing unit and target molecule.28 Therein, DNAtemplated CuNPs were demonstrated to be an excellent label-free sensing unit due to fast synthesis, excellent optical properties, and intrinsic containing isotopes, which are suitable for both fluorescence and metal stable isotopes analysis.29-34 For instance, He et al. composed a double-stranded DNA templated CuNPs system for the hydrogen peroxide related biosensing with a high sensitivity.35 Li et al. developed a histone-DNA interaction-based fluorescent bioassay with high sensitivity and stability.36 Towards ultra-sensitive detection, Lv et al. realized DNA-templated CuNPs based label4


Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


free DNA assay with inductive coupled plasma mass spectrometry (ICPMS) detection. Intrinsic copper isotopes from CuNPs were detected by ICPMS, which possess outstanding sensing capability with LOD of 4 pM.37 ICPMS has become increasingly popular in environmental and biological research in recent years as one of the preferred methods for accurate quantification, due to its one-billionth (ppt) level sensitivity, long-term signal stability, 9 orders of magnitude dynamic range and accurate quantitative capabilities traceable to primary SI units. Herein, we report a label-free CRISPR/Cas9 assay based on DNA-templated CuNPs, with merits of rapid, sensitive, and versatile for different gene. Besides, the priori in-vitro site-specific and potential off-target mutation loci detection were realized. We present a 57-bp length dsDNA substrate composed by three parts served as a model sequence. As shown in Scheme 1, except for middle green segment for complementary recognition with sgRNA and right red segment of a protospacer adjacent motif (PAM) sequence recognized by Cas9, we specifically designed the blue segment as left as s poly(ATTA) segment, which is the specific sequence to in-situ synthesize dsDNA-templated CuNPs with high efficiency.38 With biotin group modified in the 5’ terminus of complementary strand, the cleavage DNA substrate fragment could be easily washed while the rest could be captured by streptavidincoated magnetic beads. Benefit by fast in-situ synthesis of CuNPs and intrinsic mental isotopes detected by ICPMS, the proposed label-free CRISPR/Cas9 assay could realize sensitive and rapid detection with site-specific detection ability.

EXPERIMENT SECTION Materials and Instrumentations All oligonucleotides (listed in Table S1) have been purified and synthesized by Shanghai Sangon 5



Page 6 of 26

Biotechnology Co. Ltd. The T7 RNA polymerase, Taq polymerase, deoxynucleotide (dNTP) solution mix, ribonucleotide (NTP) RNAclean Kit and DEPC treated water are commercially purchased from Sangon Biotechnology Co. Ltd. as well. The Cas 9 endonuclease with reaction buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 6.5) was commercially purchased from Tolo Biotech (Shanghai, China). Besides, 3-(N-morpholino) propanesulfonic acid (MOPS) and ascorbic acid were purchased from Sigma-Aldrich (Shanghai, China). Copper sulfate anhydrous was purchased from J&K Scientific Ltd. (Beijing, China). Dynabeads M-280 Streptavidin (SA-MBs) was purchased from Thermo Fisher Scientific Inc. All the water used in Cas9 cleavage reaction was nuclease-free under DEPC treated. Besides, deionized water (18.2 MΩ cm-1) produced by a Mili-Q ultrapure system was uesd for subsequent label-free experiment. Mental stable isotopes detection was proceeded by ICPMS (NexION 350, PerkinElmer, Inc). The working conditions of ICPMS were optimized as Table S2. Energy Dispersive Spectrum (EDS) was processed by scanning electron microscopy (SEM, Hitachi, S3400). Transmission electron microscopy (TEM) with high-resolution TEM (HR-TEM) images were obtained by JEM-2010 microscope (JEOL Co., Japan) at accelerating voltage of 200 kV.

Production and Purification of sgRNA sgRNA was pre-synthesized through in vitro transcription (IVT). A 141-mer DNA template sequence, containing T7 promoter from the S. pyogenes CRIPR and trans-activating RNA sequences fused by a GAAA tetraloop linker, was pre-synthesized as the template for IVT. The sgRNA transcription reaction was proceeded with purified DNA template and T7 RNA polymerase for 3 h at 37 °C. Then RNAclean Kit was employed to purify the IVT product. The purified sgRNA was immediately used 6


Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


or stored at -80 °C. Prior to subsequent experiment, the stored sgRNA was re-folded at 95 °C for 5 min, then cooling to room temperature slowly.

Cas9 Nuclease Cleavage Assay As shown in Scheme 1, a 57-bp length dsDNA substrate composed by three parts served as a model sequence. The middle green segment was used for the complementary recognition with sgRNA. The right side of grey and red sequence consists of random grey sequence and red sequence of a protospacer adjacent motif (PAM) sequence on the noncomplementary strand, which can be recognized by Cas9. Except the same two parts in substrate, the blue sequence at left is poly(AT-TA) dsDNA segment. As dsDNA template, the introduction of poly(AT-TA) sequence can hardly influence the activity of CRIPR/Cas9 system compared to other sensing unit label. 20-nt segment of sgRNA is complementary to DNA substrate. Subsequently, sgRNA was bound with Cas9 to form Cas9/sgRNA complex, which can cleave the PAM sequence recognized DNA substrate. After the cleavage, substrate split into two parts including one with poly(AT-TA) and the other containing PAM sequence. With biotin group modified in the 5’ terminus of complementary strand, the segment containing PAM sequence and biotin group as well as remaining un-cleaved DNA substrate could be captured by SA-MBs and easily separated from the cleaved poly(AT-TA) segment. The un-cleaved poly(AT-TA) can serve as templated to in-situ synthesize CuNPs rapidly. With nitric acid digestion, the released copper ions can be detected as metal stable isotope by ICPMS. Cas9 Nuclease was pre-incubated with 30 nM sgRNA in reaction buffer for 10 min at 25 °C. Then 10 μL biotin modified substrate dsDNA (3 nM) was introduced in the system. The Cas9 cleavage reaction was conducted at 37 °C for 1 h followed by heating to 95 °C for 5 min to quench the cleavage and 7



Page 8 of 26

release dsDNA.

Label-Free Assay Procedures The Cas9 Nuclease reaction production was directly mixed with Dynabeads M-280 Streptavidin (0.1 mg mL-1 final concentration) in isometric 2X B&W buffer (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, pH 7.5) for biotin-streptavidin covalently connection. After 20 min gentle vibration, the SA-MBs captured DNA substrate can be easily magnetic separated from cleaved DNA substrate. The 5 min label-free CuNPs synthesis procedures were conducted as our previous work.30 The kinetic curve also demonstrated that 5 min cost is totally enough to generate CuNPs (Figure. S5). Briefly, followed by DIW washed twice, the SA-MBs captured DNA substrate was mixed with MOPS buffer, 1mM ascorbic acid and 50 nM copper sulfate to generate CuNPs. After washed twice, 200 μL of nitric acid (20% v/v) was introduced and digested for 10 min at 37 °C to digest the CuNPs. The final solution was diluted with DIW to 4 mL then analyzed by ICPMS.

RESULTS AND DISCUSSION IVT Based Strategy for sgRNA Synthesis In general, copper isotopes intrinsically from CuNPs were used to analyze the enzymatic activity of Cas9/sgRNA system. sgRNA, which is a specific RNA ribonucleoprotein search string for directive guiding Cas9 to cleavage site of dsDNA, was produced by IVT procedures with DNA template as Figure 1a. The DNA template sequence contains a T7 promoter, a DNA-specific guide sequence as well as remainder sequence for sgRNA designed to induce the Cas9 cleavage reaction. The IVT based strategy is widely applied to generate sgRNA rapidly. In Figure 1b, the electrophoretic bands of both 8


Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


lane 4 and lane 5 stand for the IVT produced sgRNA, while only the product in lane 4 has been treated with heat-denatured process. The clear and single electrophoretic band appears on lane 4, just because multimer of sgRNA formed as shown in lane 5, which can be easily removed by thermal denaturation before Cas9 reaction.

Structure of sgRNA Affects the Activity of Cas9 The sgRNA contains three segments: a 20-nt guide sequence, a 20-nt repeat: antirepeat duplexes region, and three tracrRNA stem loops. The guide sequence is necessary for target recognition to DNA substrate while the 30-nt repeat: antirepeat duplexes region and three tracrRNA stem loops are used for trigger the Cas9 cleavage activity. The crystal structure formed by sgRNA and Cas9 presented the helical recognition (REC) lobe of Cas9 rearranged at the same time with binding.39,40 It is an activated Cas9/sgRNA complex which is able to proceed site-specific cleavage of DNA substrate. Three tracrRNA stem loops play an important role in the crystal structure formation. Therefore, as shown in Figure 1a and 1b, we designed three sgRNA in different structure: sgRNA with one stem loop (stem loop 1), sgRNA with two stems loops (stem loop 2) and the complete sgRNA with three stem loops (stem loop 3), respectively. The polyacrylamide gel electrophoresis image demonstrated different length with the three different structures, respectively. At same conditions, Figure 1c shows that the 63Cu

ICPMS intensity decreased with the increase in stem loops. With complete three stem loops,

sgRNA is able to efficiently promote Cas9 endonuclease cleavage to dsDNA substrate, which demonstrated that the structure of tracrRNA stem loops has significant affection to form active Cas9/sgRNA complex.

9



Page 10 of 26

Performance of Label-Free Assay for Cas9 Nucleases To remove the fluorophore, radiolabel or ECL labels of DNA substrate, we replace the left part of DNA substrate with poly(AT-TA) sequence as blue segment shown in Scheme 1. Poly(AT-TA) segment located at the other end of PAM sequence, which will not influence the complementary and recognition between DNA substrate and sgRNA/Cas9 complex. Poly(AT-TA) sequence can serve as ideal template to in-situ synthesize CuNPs which intrinsically contain large amount of copper isotopes suitable for ICPMS quantification. With SA-MBs captured, remaining DNA substrate can be easily separated from segments cleavaged, which is available for indicating Cas9 nuclease activity. Considering only the existence of both Cas9 and sgRNA can lead to cleavage reaction. Figure 2a shows that one of Cas9/sgRNA missed can hardly trigger the cleavage, which demonstrating the mechanism of label-free assay of Cas9 nuclease. Several factors have been investigated for the optimal conditions. Due to dsDNA substrate serve as both target substrate of Cas9 and template of CuNPs, its concentration turned to be a crucial factor to analytical performance. Besides, random dsDNA sequence can also trigger weak synthesis of CuNPs compared to poly(AT-TA) sequence. To reduce unspecific CuNPs synthesis and increase signal to noise ratio (S/N), the concentration of dsDNA substrate is necessary. As shown in Figure 2b, despite the ICPMS intensity of Cu63 keep increasing with dsDNA concentration, the supreme S/N value appears at the concentration of 6 nM, then start to decrease. Accordingly, 6 nM dsDNA was chosen for follow-up research. The principle of in situ synthesis of CuNPs on DNA template is that Cu(II) is first reduced to Cu(I) and Cu(0) in the presence of ascorbic acid, then Cu(0) is accumulated on the DNA template. To acquire the optimal signal to noise ratio and minimize unspecific adsorption, the concentrations of Cu2+ was investigated in the first place as key factor. Different concentrations of 10


Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cu2+ was introduced after 1 mM ascorbic acid incubated with DNA substrate for 1 min. The ICPMS intensity was acquired after acid digestion. As shown in Figure 2c, 50 nM Cu2+ shows enough concentration to generate CuNPs. As for Cas9/sgRNA complex system, Cas9 reaction time as well as sgRNA concentration has also been optimized as Figure S1 and S2. 30 nM sgRNA and 60 min reaction time were picked for further experiment. Furthermore, the DNA template CuNPs was analyzed by ICPMS as copper isotope through nitric acid digestion. Hence, we investigated the digestion kinetic as shown in Figure 2d. The CuNPs could be fully transferred into ions with 10 min at 37 °C, which made it possible to realize rapid detection. Two isotopes of Cu with each abundance of 69.2% (63Cu) and 30.8% (65Cu) exist in nature. Although both isotopes of Cu are available for ICPMS detection, 63Cu

was chosen for further research for the better sensitivity than 65Cu (Fig. S3).

The DNA templated CuNPs was characterized by EDS TEM and HR-TEM images. The comparison of TEM images of SA-MBs before and after label-free CRISPR/Cas9 assay shows visible nanoparticles formed on the surface. The crystal lattice structure of CuNPs is demonstrated by HRTEM image with 0.30 nm lattice fringe spacing derived from the (110) lattice of CuNPs (Fig. 3a). And EDS spectrum confirmed the existence of Cu after synthesis (Fig. 3b and S4) as well. With the optimal conditions, the relationship between 63Cu ICPMS signal and various concentrations of Cas9 nuclease was studied. The

63Cu

ICPMS signal decreased remarkably with increasing Cas9

concentration (Fig. 3c). Due to the effect of Ostwald ripening, the CuNPs was not synthesized linearly from the crystal nucleus. As shown in Fig. 3d, a ideal linear relationship was exhibited between 63Cu signal and the logarithm of Cas9 concentration. The linear regression equation is Y=5542.7[lgX]+16590 (lgX stands for the common logarithm of Cas9 concentration with the correlation coefficient R=0.98, while Y is

63Cu

ICPMS intensity). And the limit of detection (LOD, 3σ) was 11



Page 12 of 26

determined to be 0.13 nM. The proposed label-free CRISPR/Cas9 assay was compared with series of common-used and novel reported CRISPR/Cas9 evaluation strategies. The proposed label-free assay possesses advantages of time-saving and sensitivity as shown in Table 1.

Site-Specific Detection of Single-base Change of DNA Substrate The mechanism of CRIPR/Cas9 demonstrated that the recognition and cleavage procedure are highly depended on both two DNA substrate strands respectively. The PAM-proximal sequence and core seed sequence (the first five nucleotides) on noncomplementary strand play a key role in Cas9/sgRNA complex recognition.39 Followed by recognition, the sgRNA is hybridized with complementary strand, while the HNH active site of Cas9 is cleaving the phosphodiester bond connecting the third and fourth nucleotide of complementary strand. In the complete process, there are totally three nucleic acid strands (two DNA substrate strands and sgRNA) taken part in as shown in Figure 4a. Therefore, we made a further exploration towards the influence of CRISPR/Cas9 activity with different loci of singlebase change in nucleic acid strand. All the mutant sequences are listed in Table S2. Firstly, the single-base change of core seed sequence and PAM-proximal sequence was investigated. Five mutational noncomplementary (NC) strand with constant mismatch site was designed as Figure 4b. The mismatch sites contain PAM sequence and proximal two sites. Figure 5a shows that only mismatched pair appeared inside PAM sequence led to remarkable ICPMS intensity decrease, while the mismatch pair in proximal sites has little influence approach to no mismatch strand. It demonstrated that Cas9 remains highly affinity and cleavage activity only if the complete PAM sequence is presented. Spontaneous and chemical-inducible single-base mutant is another source of mismatch. The commonhappened situation includes mismatched U-G pair41, high level of hypoxanthine42 and cytosine 12


Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


methylation43, which all connected to bioenvironment cause. So, it is important to investigate that if these DNA mutations infect CRISPR/Cas9 activity. We presented five different mutant noncomplementary strand, containing methylated cytosine, two different loci of U-G base pair and two loci of I-C base pair at opposite sides of PAM sequence. As shown in Figure 4c, it’s not hard to see that the designed mutant noncomplementary strands have little influence to the activity of CRISPR/Cas9, similar to no mutant group. The possible reason might be these kinds of structural distortions is not enough to generate steric hindrance to prevent Cas9 cleavage26. By all the above experiments, the single-base change in noncomplementary strand outside of PAM sequence can hardly impact the effect of CRISPR/Cas9 system. Besides, the other complementary strand is in charge of hybridization with Casy9/sgRNA and cleaved by the HNH site of Cas9. Theoretically, single-base change in cleavage site might interfere the CRISPR/Cas9 activity. In order to verify that, we designed five complementary strands with different mismatch site. Among them, the mismatch site of C2 is located at the third nucleotide of the complementary strand-sgRNA hybrid. As shown in Figure 4d, all the mismatch sequence except C2 exhibit almost the same cleavage efficiency as no mismatch group, including C3, the mismatch site located at the original complementary area against PAM sequence. The C2 shows a relatively decreased 63Cu ICPMS intensity, which might be because the spatial distortions induced by nucleotide base mismatch at cleavage site influence the activity of HNH site of Cas9. The result demonstrated that the proposed label-free Cas9 assay could detect single base change at cleavage site. However, it is not accordance when the single-base change happened in sgRNA. Hybridization of sgRNA and complementary strand is the key initial period of CRISPR/Cas9 system. Noncorresponding sgRNA means off-target DNA substrate to the system. To verify the capability of 13



Page 14 of 26

distinguishing off-target substrate, three single-base changed sgRNA was introduced as Figure 4e. The changed site distributes inside the complementary part of core seed sequence. Compared to the corresponding sgRNA, the cleavage efficiency remarkably decreased with different level. The mismatch site more faraway from PAM sequence, the less the cleavage efficiency was influenced. Still, compared to control group, there have been nonspecific cleavage in off-target system with unexpected sites. With different off-target substrate design, the proposed label-free Cas9 assay could detect potential undersigned substrate cleavage sites, further avoid off-target effects.

Conclusion In summary, we have presented a DNA templated CuNPs based label-free CRIPR/Cas9 assay enable site-specific detection. The proposed method possesses several inspiring features for in-vitro evaluation of CRISPR/Cas9 system as follows: (1) the proposed label-free strategy leaves out common-used labels including fluorophore/quencher pairs, electrochemiluminescence label and radiolabels, providing a time-saving analysis and avoid possible disturbance to Cas9 activity, suitable for high-throughput in-vitro analysis. (2) A nano-molar level of LOD is achieved, for solving the bottleneck of single-turnover enzyme feature of Cas9 nuclease. (3) The site-specific detection of DNA substrate is realized by combining site-specific CRISPR/Cas9 cleavage and in-situ CuNPs synthesis. Single-base change in complementary strand and sgRNA could be successfully detected. With predesign according to different gene sequence, our method could apply in the cast majority of off-target probability detection. On the basis of the advantages above, the proposed DNA templated CuNPs based label-free CRIPR/Cas9 assay might provide a novel priori analysis strategy towards leading further in-vivo CRISPR/Cas9 genome editing research. 14


Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ASSOCIATED CONTENT Supporting Information. This Supporting Information is available free of charge on the ACS Publication website at DOI: xxxxxxx DNA sequence, mutant sequence design, operating conditions of ICPMS, additional and condition optimizations and EDS spectra of original MBs.

ACKNOWLEDGMENTS The National Natural Science Foundation of China is appreciatively acknowledged (No. 21505008, &21575093). This work is also supported by the Recruitment Program of Global Experts of Sichuan Province (No. 903), Sichuan Science and Technology Program (19CXRC0047), and the Fundamental Research Funds for the Central Universities. Dr. Chunxia Wang from College of Chemistry, Sichuan University, and Dr. Shanlin Wang and Dr. Shuguang Yan from Analytical & Testing Center, Sichuan University, are gratefully thanked for technical assistance.

15



Page 16 of 26

Reference (1) Brouns, S. J. J.; Jore, M. M.; Lundgren, M.; Westra, E. R.; Slijkhuis, R. J. H.; Snijders, A. P. L.; Dickman, M. J.; Makarova, K. S.; Koonin, E. V.; van der Oost, J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008, 321, 960-964. (2) Horvath, P.; Barrangou, R. CRISPR/Cas, the Immune System of Bacteria and Archaea. Science 2010, 327, 167-170. (3) Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A. Charpentier E. A programmable dual-RNA– guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816-821 (4) Ran, F. A.; Cong, L.; Yan, W. X.; Scott, D. A.; Gootenberg, J. S.; Kriz, A. J.; Zetsche, B.; Shalem, O.; Wu, X. B.; Makarova, K. S.; Koonin, E. V.; Sharp, P. A.; Zhang, F. In vivo genome editing using Staphylococcus aureus Cas9. Nature 2015, 520, 186-U198. (5) Sun, W. J.; Ji, W. Y.; Hall, J. M.; Hu, Q. Y.; Wang, C.; Beisel, C. L.; Gu, Z. Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing. Angew. Chem.-Int. Edit. 2015, 54, 12029-12033. (6) Shah, A. N.; Davey, C. F.; Whitebirch, A. C.; Miller, A. C.; Moens, C. B. Rapid reverse genetic screening using CRISPR in zebrafish. Nat. Methods 2015, 12, 535 (7) Li, Y.; Teng, X. C.; Zhang, K. X.; Deng, R. J.; Li, J. H. RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing. Anal. Chem. 2019, 91, 3989-3996. (8) Zhang, K. X.; Deng, R. J.; Teng, X. C.; Li, Y.; Sun, Y. P.; Ren, X. J.; Li, J. H. Direct Visualization of Single-Nucleotide Variation in mtDNA Using a CRISPR/Cas9-Mediated Proximity Ligation Assay. J. Am. Chem. Soc. 2018, 140, 11293-11301. (9) Ran, F. A.; Hsu, P. D.; Lin, C. Y.; Gootenberg, J. S.; Konermann, S.; Trevino, A. E.; Scott, D. A.; Inoue, A.; Matoba, S.; Zhang, Y.; Zhang, F. Double nicking by RNA-Guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013, 154, 1380-1389. (10) Pattanayak, V.; Lin, S.; Guilinger, J. P.; Ma, E. B.; Doudna, J. A.; Liu, D. R. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotech. 2013, 31, 839. (11) Wu, X. B.; Scott, D. A.; Kriz, A. J.; Chiu, A. C.; Hsu, P. D.; Dadon, D. B.; Cheng, A. W.; Trevino, A. E.; Konermann, S.; Chen, S. D.; Jaenisch, R.; Zhang, F.; Sharp, P. A. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotech. 2014, 32, 670. 16


Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Akcakaya, P.; Bobbin, M. L.; Guo, J. A.; Malagon-Lopez, J.; Clement, K.; Garcia, S. P.; Fellows, M. D.; Porritt, M. J.; Firth, M. A.; Carreras, A.; Baccega, T.; Seeliger, F.; Bjursell, M.; Tsai, S. Q.; Nguyen, N. T.; Nitsch, R.; Mayr, L. M.; Pinello, L.; Bohlooly, Y. M.; Aryee, M. J.; Maresca, M.; Joung, J. K. In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 2018, 561, 416-419. (13) Yin, H.; Song, C. Q.; Suresh, S.; Kwan, S. Y.; Wu, Q.; Walsh, S.; Ding, J.; Bogorad, R. L.; Zhu, L. J.; Wolfe, S. A.; Koteliansky, V.; Xue, W.; Langer, R.; Anderson, D. G. Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. Nat. Chem. Biol. 2018, 14, 311-316. (14) Fu, Y. F.; Sander, J. D.; Reyon, D.; Cascio, V. M.; Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotech. 2014, 32, 279-284. (15) Doench, J. G.; Hartenian, E.; Graham, D. B.; Tothova, Z.; Hegde, M.; Smith, I.; Sullender, M.; Ebert, B. L.; Xavier, R. J.; Root, D. E. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotech. 2014, 32, 1262-1130. (16) Kiani, S.; Chavez, A.; Tuttle, M.; Hal, R. N.; Chari, R.; Ter-Ovanesyan, D.; Qian, J.; Pruitt, B. W.; Beal, J.; Vora, S.; Buchthal, J.; Kowal, E. J. K.; Ebrahimkhani, M. R.; Collins, J. J.; Weiss, R.; Church, G. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 2015, 12, 1051-1054. (17) Bassett, A. R.; Tibbit, C.; Ponting, C. P.; Liu, J. L. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 2013, 4, 220-228. (18) Raper, A. T.; Stephenson, A. A.; Suo, Z. Sharpening the Scissors: Mechanistic Details of CRISPR/Cas9 Improve Functional Understanding and Inspire Future Research. J. Am. Chem. Soc. 2018, 140, 11142-11152. (19) R Richardson, C. D.; Ray, G. J.; DeWitt, M. A.; Curie, G. L.; Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotech. 2016, 34, 339. (20) Zhang, K.; Deng, R.; Li, Y.; Zhang, L.; Li, J. Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification. Chem. Sci. 2016, 7, 4951-4957. (21) Huang, M.; Zhou, X.; Wang, H.; Xing, D. Clustered regularly interspaced short palindromic repeats/Cas9 triggered isothermal amplification for site-specific nucleic acid detection. Anal. Chem. 2018, 90, 2193-2200. (22) Bell, C. C.; Magor, G. W.; Gillinder, K. R.; Perkins, A. C. A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing. BMC Genomics 2014, 15, 1002. (23) Mekler, V.; Minakhin, L.; Semenova, E.; Kuznedelov, K.; Severinov, K. Kinetics of the CRISPR-Cas9 17



Page 18 of 26

effector complex assembly and the role of 3'-terminal segment of guide RNA. Nucleic Acids Res. 2016, 44, 28372845. (24) Mekler, V.; Minakhin, L.; Severinov, K. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation. Proc Natl Acad Sci U S A 2017, 114, 5443-5448. (25) Seamon, K. J.; Light, Y. K.; Saada, E. A.; Schoeniger, J. S.; Harmon, B. Versatile high-throughput fluorescence assay for monitoring Cas9 activity. Anal. Chem. 2018, 6913-6921. (26) Liu, W.; Yu, H.; Zhou, X.; Xing, D. In vitro evaluation of CRISPR/Cas9 function by an electrochemiluminescent assay. Anal. Chem. 2016, 88, 8369-8374. (27) Cox, K. J.; Subramanian, H. K. K.; Samaniego, C. C.; Franco, E.; Choudhary, A. A universal method for sensitive and cell-free detection of CRISPR-associated nucleases. Chem. Sci. 2019, 10, 2653-2662. (28) Luo, X. L.; Davis, J. J. Electrical biosensors and the label free detection of protein disease biomarkers. Chem. Soc. Rev. 2013, 42, 5944-5962. (29) Liu, R.; Wang, C.; Hu, J.; Su, Y.; Lv, Y. DNA-templated copper nanoparticles: Versatile platform for labelfree bioassays. TrAC Trend. Anal. Chem. 2018, 105, 436-452. (30) Hu, J.; Wang, C.; Liu, R.; Su, Y.; Lv, Y. Poly(thymine)-CuNPs: Bimodal methodology for accurate and selective detection of TNT at sub-PPT Levels. Anal. Chem. 2018, 90, 14469-14474. (31) Liu, R.; Zhang, S.; Wei, C.; Xing, Z.; Zhang, S.; Zhang, X. Metal stable isotope tagging: renaissance of radioimmunoassay for multiplex and absolute quantification of biomolecules. Accounts Chem. Res. 2016, 49, 775783. (32) Wang, Y.; Cui, H. Y.; Cao, Z. J.; Lau, C. W.; Lu, J. Z. Additive and enhanced fluorescence effects of hairpin DNA template-based copper nanoparticles and their application for the detection of NAD(+). Talanta 2016, 154, 574-580. (33) Sun, F.; You, Y.; Liu, J.; Song, Q. W.; Shen, X.; Na, N.; Ouyang, J. DNA three-way junction for differentiation of single-nucleotide polymorphisms with fluorescent copper nanoparticles. Chem. Eur. J. 2017, 23, 6979-6982. (34) Liu, R.; Hu, J.; Chen, Y.; Jiang, M.; Lv, Y. Label-free nuclease assay with long-term stability. Anal. Chem. 2019, 91, 8691-8696. (35) Chen, J. Y.; Ji, X. H.; He, Z. K. Smart composite reagent composed of double-stranded DNA-templated copper nanoparticle and SYBR Green I for hydrogen peroxide related biosensing. Anal. Chem. 2017, 89, 3988-3995. 18


Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36) Lian, J. Y.; Liu, Q.; Jin, Y.; Li, B. X. Histone-DNA interaction: an effective approach to improve the fluorescence intensity and stability of DNA-templated Cu nanoclusters. Chem. Comm. 2017, 53, 12568-12571. (37) Liu, R.; Wang, C.; Xu, Y.; Hu, J.; Deng, D.; Lv, Y. Label-free DNA sssay by metal stable isotope detection. Anal. Chem. 2017, 89, 13269-13274. (38) Song, Q. W.; Shi, Y.; He, D. C.; Xu, S. H.; Ouyang, J. Sequence-dependent dsDNA-templated formation of fluorescent copper nanoparticles. Chem. Eur. J. 2015, 21, 2417-2422 (39) Jiang,F.; Zhou, K; Ma, l.; Gressel S.; Doudna J. A. A Cas9–guide RNA complex preorganized for target DNA recognition. Science 2015, 348, 1477−1481. (40) Nishimasu, H.; Ran, F. A.; Hsu, P. D.; Konermann, S.; Shehata, S. I.; Dohmae, N.; Ishitani, R.; Zhang, F.; Nureki, O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 2014, 156, 935-949. (41) Wickramasinghe, S. N.; Fida, S. Bone-marrow cells from vitamin-B-12-deficient patients misincorporate uracil into DNA. Blood 1994, 83, 1656-1661. (42) Spencer, J. P. E.; Whiteman, M.; Jenner, A.; Halliwell, B. Nitrite-induced deamination and hypochloriteinduced oxidation of DNA in intact human respiratory tract epithelial cells. Free Radic. Biol. Med. 2000, 28, 10391050. (43) Bestor, T. H.; Verdine, G. L. DNA methyltransferases. Curr. Opin. Cell Biol. 1994, 6, 380-389. (44) Nelson, P. S.; Shermangold, R.; Leon, R. A New and vasitile reagent for incoporating multiple primary alaphatic-amines into synthetic oligonucleotides. Nucleic Acids Res. 1989, 17, 7179-7186. (45) Liu, W. P.; Zhou, X. M.; Xing, D. Rapid and reliable microRNA detection by stacking hybridization on electrochemiluminescent chip system. Biosens. Bioelectron. 2014, 58, 388-394. (46) Stephenson, A. A.; Raper, A. T.; Suo, Z. Bidirectional degradation of DNA cleavage products catalyzed by CRISPR/Cas9. J. Am. Chem. Soc. 2018, 140, 3743-3750.

19



For TOC only

20


Page 20 of 26

Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. The principle of the label free CRISPR/Cas9 assay.

21



Page 22 of 26

Figure 1. In vitro transcription of sgRNA. (a) Schematic of IVT and structure of sgRNA. (b) Polyacrylamide gel electrophoresis of IVT products: Lane 1, marker; Lane 2, sgRNA with one stem loop; Lane 3, sgRNA with two stem loops; Lane 4, sgRNA with complete three stem loops; Lane 5, sgRNA without heat denatured process. (c) Relationship between structure of sgRNA and Cas9 cleavage activity. (sgRNA 30 nM and dsDNA substrate 6 nM)

22


Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Experiment conditions optimizations. (a) 63Cu ICPMS intensity with different reaction conditions at the existence or absence of sgRNA and Cas9 nuclease. (b) 63Cu ICPMS intensity and signal to noise ratio with various amount of dsDNA substrate from 0-20 nM. Optimizations of experiment conditions: 63Cu ICPMS intensity with (c) different Cu2+ concentrations (0, 10, 25, 50, 70 and 100 nM respectively). (d) kinetic curves of nitric acid digestion (sgRNA 30 nM, dsDNA substrate 6 nM and Cas9 500nM).

23



Page 24 of 26

Figure 3. Characterizations of CuNPs and calibration curve. (a) HR-TEM and comparison of TEM images of DNA templated CuNPs on SA-MBs. The bottom half belongs to SA-MBs captured DNA substrate after CuNPs synthesis, while the upper one shows original SA-MBs. (b) EDS of SA-MBs captured DNA substrate after label-free CRISPR/Cas9 assay. (c) Relationship between 63Cu ICPMS signal and several Cas9 concentrations. (d) Calibration curve between lg(Cas9 nuclease concentration) and 63Cu signal.

24


Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Site-specific detection with proposed label-free CRISPR/Cas9 assay, containing detailed sequence design with mutant site highlighted in yellow and 63Cu ICPMS intensity of corresponding single-base change sequence. (a) Schematic of sgRNA-DNA substrate heteroduplex. PAM sequence on noncomplementary strand colored with dark red. Cleavage sites are marked by bright red triangles in each of substrate strands. (b) The single-base mismatch sequences of noncomplementary strand. (c) The single-base mutant of noncomplementary strand. (d) The single-base mismatch sequence of complementary strand. (e) The single base mismatch of sgRNA. Control groups: (a), (b), (c) dsDNA substrate 6 nM with sgRNA 30 nM and no Cas9. (d) dsDNA substrate 6 nM without sgRNA or Cas9.

25



Page 26 of 26

Table 1. Comparison of analytical performance of proximately reported CRISP/Cas9 evaluation methods Label Process Time Cost

LOD

Sensing Unit

Analytical Technology

Detection Time Cost

Fluorophore/ quencher pair

Fluorescence

＜5 min

4.5 hours

＜30 nM

25,44

Ru(bpy)32+ label

Electrochemiluminescence

＜5 min

＞12 hours

1 nM

26,45

Fluorescence

4 hours

5 nM

27

Gel electrophoresis

3 hours

NG

46

40 min

10 pM

20

35 min

0.13 nM

This work

DFHBI-RNA fluorescent complex 32P

radiolabeled DNA substrate

dsDNA binding dye

Fluorescence-based

EvaGreen®

isothermal amplification

Label-free CuNPs

ICPMS

*NG: Not given

26


NG*

(3σ)

Reference

Cas9 Assay for Site-Specific ... - ACS Publications

Recommend Documents