Subscriber access provided by Northern Illinois University
Letter
Rapid in vitro Evaluation of CRISPR/Cas9 Function by an Electrochemiluminescent Assay Weipeng Liu, Huiheng Yu, Xiaoming Zhou, and Da Xing Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02338 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 9, 2016
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 free 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 accessible to all readers and 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.
Analytical Chemistry 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 6
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
Rapid in vitro Evaluation of CRISPR/Cas9 Function by an Electrochemiluminescent Assay Weipeng Liu, Huiheng Yu, Xiaoming Zhou*, Da Xing* MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China * To whom correspondence should be addressed, E-mail:
[email protected], correspondence may also be addressed to Email:
[email protected]; Tel: +86-20-85210089; Fax: +86-20-85216052 ABSTRACT: The CRISPR/Cas9 system is a revolutionary genome-editing tool that enables targeted and efficient gene knockouts. However, the off-target effects and loci-dependent enzyme activity limit its uses on the field of research and treatment. In this study, we designed a convenient and sensitive in vitro test method, which was based on Electrochemiluminescence (ECL) technology for evaluating cleavage activity of CRISPR/Cas9 system. It was find that Cas9 can tolerate some common genetic modifications to its target DNA. It was also find that target DNA/sgRNA with single-base mismatch and UV damages of target DNA resulted in significantly reduction of Cas9 cleavage efficiency. Comparing with traditional method, the proposed method reduced the evaluation time from weeks to 2 hours. Therefore, our study provides a versatile in vitro method for a priori analysis of CRISPR/Cas9 system and highlights the potential to guide in vivo genome editing.
Introduction The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas systems were firstly found in the bacterial and archaeal as an RNA-directed defense system which can degrade invading DNA of viruses and plasmids1-3. Among them, the type Ⅱ CRISPR-Cas system from Streptococcus pyogenes was merely needed the Cas9 nuclease and an engineered single guide RNA (sgRNA)4. In recent years, due to the Cas9-mediated site-specific DNA break is easily designed and highly effective, researches have rapidly extended it to the genome-editing fields such as studying pathogenesis and treating diseases5-7. Previous studies demonstrated that the Cas9 can specifically break the designed location of target DNA, but some mistaken cleavage events can still happen8-12. It means that there may be several off-target sites for a given Cas9/sgRNA complex in a genome-editing assay. Off-target effect may lead to genetic disruption at undesigned loci, and may be dangerous to health of organism. Therefore, the utility of CRISPR-Cas9 system may be compromised by the off-target activity in research and disease treatment. Generally, researches addressed this problem by using the CruiserTM enzyme or T7 endonuclease based PCR assay after the target cell transfections and cultures. This process will take 8-14 days, and will be complicated by the laborious procedures13. Cho’s team optimized the method by using deep sequencing technology to further improve the detecting sensitivity of off-target mutations9. However, in these methods, the assessment steps cannot be performed until the Cas9-mediaed genome-editing step is finished. In recent years, radioisotope-labeled gel electrophoreses has become a standard priori method for analyzing off-target effects. However, the invasiveness of radioisotope poses a serious health risk for researches. Therefore, conventional methods
used to evaluate on-target CRISPR/Cas9 events are complex, time consuming and risky. More importantly, researchers found that different genomic loci showed remarkably different efficiencies of Cas9 cleavage5. Therefore, there is an urgent need to find a simple, convenient and highly efficient method to evaluate the specificity and activity of Cas9 platform before in vivo assay. Electrochemiluminescence (ECL) is a significant and powerful tool in analytical and detection fields with high sensitivity, wide dynamic range, and low cost14,15. Meanwhile, the ECL active species Ru(bpy)32+ is a small molecule which can easily be labeled on nucleic acid and causes minimal disturbance to enzymatic activity. In our previous work, we successfully analyzed the protein kinase activities using ECL biosensor and obtained a relatively low detection limit16. Furthermore, the surface-functionalized magnetic beads (MB) have been widely used in the fields of nucleic acids purification, drug load and immunoassay due to its ability of selectivity binding and purification of target analytes. Therefore, the MB-based ECL technology is a promising method for the detection of enzyme activity and analysis of substrate specificity. In this paper, we first reported a rapid, sensitive and inexpensive ECL-based in vitro test method for evaluation of CRISPR-Cas9 genome editing system. We can predict the potential off-target mutations locus and the efficiency of target DNA double-strand breaks before deliver CRISPR/Cas9 plasmid into receptor cells. The basic principle of this detection method is shown in Figure 1. In this strategy, a 50-bp length dsDNA is used as model sequence. The complementary strand of the target dsDNA was labelled with a biotin group at the 5' terminus and a Ru(bpy)32+ group at the 3’ terminus. The synthesis of Ru(bpy)32+-NHS ester and the labeling of TBRDNA were completed as described previously in our team
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
reports.17 The target DNA contains an adjacent PAM sequence on the non-complementary strand which is recognized by Cas9. A 20-nt guide sequence of the sgRNA is complementary to the target DNA. Then, sgRNA was bound with Cas9 to form a functional complex, which can cleave the recognized DNA. Due to the target DNA labelled with a biotin, it will covalently connect with the streptavidin-coated magnetic beads, and the cleaved DNA can be easily washed thoroughly using a magnetic separator. The proposed approach reduces the test time of Cas9 system from a matter of days or weeks to some hours.
Figure 1. Experimental principle of ECL-based strategy for the detection of Cas9 activity.
Experimental Section Materials All oligo nucleotides (the sequences are listed in Table S1) were synthesized and purified by Generay Biotech Co., Ltd (Shanghai, China). The Taq polymerase, T7 RNA polymerase, their corresponding buffer, ribonucleotide (NTP) solution mix, deoxynucleotide (dNTP) solution mix and RNase inhibitor all were purchased from Sangon Biological Engineering and Technology & Services Co., Ltd (Shanghai, China). The Cas9 endonuclease and corresponding buffer were purchased from Tolo Biotech (Shanghai, China). The tripropylamine (TPA) and other chemicals to synthesize the Ru(bpy)32+-NHS were purchased from Sigma (St. Louis, MO, USA). The water (≥18.2 MΩ cm) used in this research was generated from a Milli-Q water-purification system (MA, USA). Production and purification of sgRNA The DNA template of sgRNA was produced by fill-in PCR. Briefly, PCR was performed with merely two long primers: (1) a 65-nt customized oligonucleotide containing the T7 promoter (TAATACGACTCACTATA) and the 20-nt sgRNA DNAtargeting sequence; (2) an 80-nt reverse oligonucleotide encoding the 3’ tail sequence of sgRNA. A 20-nt length of these two primers was completing complementary. PCR was carried out in a thermal cycler with the following protocol: 95 °C for 2 min; 35 cycles of 95 °C for 20 s, 60 °C for 30 s and 72 °C for 15 s; and a final step of 72 °C for 10 min. The PCR product (125-bp) was purified using SanPrep Column PCR Product Purification Kit and was used as a template for a T7 RNA polymerase-mediated transcription reaction. The sgRNA transcription reaction was performed with purified DNA template for 6 hours at 30 °C, and the product was purified by RNAclean Kit (Tiangen). The purified sgRNA was used immediately for DNA cleavage assay or frozen at -80 °C. Target DNA cleavage assay Cas9 Nuclease (30 nM final concentration) was firstly incubated with the sgRNA (30 nM) in reaction buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 6.5) in a total volume of 27 µl. The mixture was pre-incubated 10
Page 2 of 6
min at 25 °C. Then, 3 µl target DNA (3 nM final concentration) was added in the mixture sample at 37 °C for 1 hour. Reactions were stopped by heating to 94 °C for 5 min.
Results and Discussion A Fill-in PCR-based strategy for generation of sgRNAs In this research, we evaluate the enzymatic activity of Cas9/sgRNA ribonucleoprotein by MB-based ECL detection. Cas9 will be guided to specifically cleave target dsDNA by a RNA search string (namely sgRNA). As shown in Figure 2A, the sgRNAs were synthesized by transcription of a dsDNA which produced by Fill-in PCR (Figure 2B) and contain a T7 RNA polymerase binding site, a DNA-specific guide sequence and a remainder sequence for sgRNA that necessary to trigger the Cas9 cleavage activity. In conclusion, it is a modular method to synthesize sgRNA. We can rapidly produce sgRNAs at different sequences for targeting various genes, or even at different length to research the activity of Cas9/sgRNA on structural flexibility. In Figure 2C, a single clear electrophoretic band, which represent sgRNA, appeared on the lane 2. However, the purified produce (lane 3) displayed two bands. This is because the sequence of sgRNA can result to form dimmer and higher multimer, and that can be resolved by thermal denaturation before Cas9 cleave experiment (lane 4).
Figure 2. In vitro transcription of sgRNA. (A) Fill-in PCR-based strategy for synthesis of sgRNA. (B) Agarose gel electrophoresis of Fill-in PCR product. (C) Polyacrylamide gel electrophoresis of T7 RNA polymerase product, namely sgRNA. Lane 1: Marker; Lane 2: sgRNA; Lane 3: purified sgRNA; Lane 4: purified and heat-denatured sgRNA.
Length of sgRNA affects the activity of Cas9 The sgRNA was fused with the 3’ terminal of crRNA and 5' terminal of tracrRNA using an artificial tetraloop sequence. It consist of three segments: a 20-nt guide sequence, a 30-nt repeat:anti-repeat duplexes region and three tracrRNA stem loops. The crystal structure of sgRNA:Cas9 indicated that the helical recognition (REC) lobe of Cas9 undergoes significant rearrangement during sgRNA binding18,19. Therefore, sgRNA may be necessary for Cas9 nuclease to form a target DNAbinding competent conformation. Herein, we design three sgRNAs with one stem loop (sgRNA (+48)), two stem loops (sgRNA (+67)) and three stem loops (sgRNA (+85)), respectively (Fig. 3A). As shown in Fig. 3B, we observed low ECL responses when the guide RNAs were sgRNA (+67) and sgRNA (+85), which means that they can efficiently direct Cas9 endonuclease cleavage of target dsDNA. On the contrary, sgRNA (+48) only leads to a weak ECL signal intensity declined comparing with control group. These phenomena suggested that the tracrRNA tail made significant 2 contributions to form an activated Cas9 conformation.
ACS Paragon Plus Environment
Page 3 of 6
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
CRISPR/Cas9 system for cleaving non-target dsDNA Previous researches have shown that the first five nucleotides (core seed sequence) of the PAM-proximal sequence of target dsDNA are the most important segment for Cas9 recognition, because the core seed sequence is the initial binding site with the target DNA and then triggering additional conformational change of Cas9 to complete pairing between the full guide sequence and the target dsDNA20. Therefore, in this paper, we laid a strong emphasis on the research of the activity of Cas9 when the core seed sequence or corresponding region of sgRNA has mutations.
Figure 3. The length of sgRNA affects the Cas9 cleavage activity. (A) The sequences of different sgRNAs. (B) The ECL intensity of different sgRNAs.
We introduced five mutational non-complementary (NC) strands and an incomplete NC strand which only contain PAM sequence and 3' region into the CRISPR/Cas9 system. As
shown in Fig. 4B, though the target dsDNA exists mismatch pairs and changes in helical geometry, the cleavage activity of Cas9 have little influence. Meanwhile, when the incomplete NC strand hybridized with complementary strand, Cas9 also can recognize and cleave the hybrid. Therefore, we believed that Cas9 can binds with high affinity to target dsDNA with NC strand mutations when the PAM sequence is presented. In order to evaluate whether Cas9/sgRNA platform could effectively distinguish off-target dsDNA, five noncorresponding sgRNAs (sgRNA MM1-5) with a single base difference were chosen (Fig. 4C). The addition of noncorresponding sgRNAs led to obvious weaker ECL signal declined compared with the strong signal change from incubation with the corresponding sgRNA (Fig. 4D). The efficiency of cleavage can be reduced to 11% by using sgRNA MM-1, but up to 33% by using sgRNA MM-4. This data indicated that some undesigned sites can also lead to nonspecific cleavage. Therefore, our method could help to find out potential false DNA cleavage sites and hence avoid off-target effects in CRISPR/Cas9 genome editing system. CRISPR/Cas9 system for cleaving single-base change dsDNA In a genome, spontaneous or chemical-inducible single-base changes are frequently happening. If cells lack of folate, dUMP will accumulate and be synthesized into DNA, which would lead to create a mismatched U-G pair20. When the healthy cells been exposed to nitrite, the level of hypoxanthine will significant increase and would be observed in DNA sequence21. Meanwhile, some nucleic acid base can be covalently attached a methyl group at the 5' carbon of cytosine, which affected the function of genes22. Therefore, it is necessary to evaluate whether these DNA changes will interfere with the CRISPR/Cas9 system. In this experiment, we designed a methylated target DNA, two U-G base pair target DNAs and two I-C base pair target DNAs (Fig. 4E). As shown in Fig. 4F, these variation of base have little impact on the endonuclease activity of Cas9. We conclude that the structural distortions of variation target DNA cannot provide an enough physical impediment to prevent Cas9-mediated cleavage.
Figure 4 Single-nucleotide specificity of Cas9. (A) The single-base mismatch sequences of target DNA (noncomplementary strand). (B) The ECL intensity of different products in the presence of single-base mismatch target dsDNA (C) The mismatch sequences of sgRNAs. (D) The ECL intensity of different products in the presence of mismatch sgRNAs. (E) The single-base change sequence of target dsDNA. (F) The ECL intensity of different products in the presence of single-base change target dsDNA.
3
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
Therefore, in the design of CRISPR/Cas9 system, it is suggested that special consideration of these single-base changes in genome is not needed. Ultraviolet radiation (UV) plays an important role in the DNA damage and cell death, and it can introduce covalent bonds between two adjacent thymine bases or pyrimidine dimer between two ssDNA. In order to simulate this phenomenon, we treated the target dsDNA with ultraviolet light (254 nm, 470 µw/cm2). As shown in Fig. 5(A), with the increase of ultraviolet irradiation time, a slowly rising ECL signal will be observed, which indicated that UV-induced DNA damage would hinder the sgRNA/Cas9 recognition causing by the distortion of double helix structure. Meanwhile, the ECL signal of control groups show a downward trend, because UV can also break the target DNA and damage the Ru(bpy)32+ group.
Page 4 of 6
of Cas9 nuclease (from 100 pM to 30 nM). (B) Linear correlation of mean ECL intensity vs. Cas9 concentration.
Conclusion In summary, we have developed a novel ECL-based in-vitro evaluation system for CRISPR-Cas9. Our method emphasizes finding ways to avoid the off-target effect, and to provide the cleavage efficiency of different target location quantitatively. Therefore, we can high-efficiently evaluate of the Cas9induced target DNA double-strand breaks. Compared with traditional Cas9 targeting efficiency detection methods, our detection system could fleetly predict the probability of offtarget events and effectively overcome the poor sensitivity of gel electrophoresis. Moreover, due to the target DNAs and sgRNAs are ease of modification, design and synthesis, our system could almost cover all different type of genes. Based on advantages above, the present method might be provided as a practical priori approach to guide in-vivo Cas9-based genome editing in the further research.
AUTHOR INFORMATION Corresponding Author
Figure 5 The UV light affects the Cas9 cleavage activity. (A) Effect of ultraviolet irradiation time on the ECL intensity. (B) The percent of Cas9-mediated cleavage vs ultraviolet irradiation time increase.
* Da Xing, PhD, Professor E-mail address:
[email protected] Xiaoming Zhou, PhD, Professor E-mail address:
[email protected] Tel: +86-20-85210089; Fax: +86-20-85216052
ACKNOWLEDGMENT The Cas9 activity analysis via the designed system At last, we investigated the sensitivity of our designed ECL detection system. Various concentrations of Cas9 (30 nM, 15 nM, 7.5 nM, 3.75 nM, 1nM and 100 pM) were added to a series of samples containing 3 nM target dsDNA. Fig. 6A showed the relationship between the ECL intensity of the target dsDNA and the concentration of Cas9 nuclease. With the addition of Cas9,
the ECL intensity is declining. However, when the ratio of the Cas9 to target dsDNA is higher than 5:1, the signal intensity has no obvious change. Furthermore, the ECL intensity is found to be linear with Cas9 concentration from 1 nM to 15 nM with a linear correlation coefficient (R2) of 0.98656 (Fig. 6B). The limit of detection (LOD) for the Cas9 detection is approximately 1 nM (three times signal-to-noise ratio). Therefore, the present method was superior to the traditional gel electrophoresis based method in term of sensitivity. Meanwhile, we found that when the mole ratio of Cas9 and target DNA is above 5:1, the cleavage efficiency comes into platform stage.
Figure 6 The sensitivity and detection range of designed ECL detection system. (A) ECL intensity of the different concentration
This work was supported by the National Natural Science Foundation of China [21475048], the National Basic Research Program of China [2010CB732602], the National Science Fund for Distinguished Young Scholars of Guang-dong Province [2014A030306008] and the Program of the Pearl River Young Talents of Science and Technology in Guangzhou, China [2013J2200021]. and the project of Guangzhou science and technology plan [201508020003]
ASSOCIATED CONTENT Supporting Information. Further experimental details including magnetic bead ECL detection of Cas9 activity and the sequences of oligo nucleotides (as shown in Table S1).
REFERENCES (1) Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. Science 2007, 315, 1709-1712. (2) Brouns, S. J.; Jore, M. M.; Lundgren, M.; Westra, E. R.; Slijkhuis, R. J.; Snijders, A. P.; Dickman, M. J.; Makarova, K. S.; Koonin, E. V.; Van Der Oost, J. Science 2008, 321, 960964. (3) Horvath, P.; Barrangou, R. Science 2010, 327, 167-170. (4) Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. Science 2012, 337, 816-821. (5) Bassett, A. R.; Tibbit, C.; Ponting, C. P.; Liu, J. L. Cell Rep. 2013, 4, 220-228. (6) Sun, W.; Ji, W.; Hall, J. M.; Hu, Q.; Wang, C.; Beisel, C. L.; Gu, Z. Angew. Chem. Int. Ed. Engl. 2015, 54, 1202912033. (7) Moreno-Mateos, M. A.; Vejnar, C. E.; Beaudoin, J. D.; Fernandez, J. P.; Mis, E. K.; Khokha, M. K.; Giraldez, A. J. 4 Nat. Methods 2015, 12, 982-988.
ACS Paragon Plus Environment
Page 5 of 6
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
(8) Hsu, P. D.; Scott, D. A.; Weinstein, J. A.; Ran, F. A.; Konermann, S.; Agarwala, V.; Li, Y.; Fine, E. J.; Wu, X.; Shalem, O.; Cradick, T. J.; Marraffini, L. A.; Bao, G.; Zhang, F. Nat. biotechnol. 2013, 31, 827-832. (9) Cho, S. W.; Kim, S.; Kim, Y.; Kweon, J.; Kim, H. S.; Bae, S.; Kim, J. S. Genome Res. 2014, 24, 132-141. (10) Pattanayak, V.; Lin, S.; Guilinger, J. P.; Ma, E.; Doudna, J. A.; Liu, D. R. Nat. Biotechnol. 2013, 31, 839-843. (11) O'Geen, H.; Henry, I. M.; Bhakta, M. S.; Meckler, J. F.; Segal, D. J. Nucleic Acids Res. 2015, 43, 3389-3404. (12) Wu, X.; 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. Nat. Biotechnol. 2014, 32, 670-676. (13) Ran, F. A.; Hsu, P. D.; Wright, J.; Agarwala, V.; Scott, D. A.; Zhang, F. Nat. protoc. 2013, 8, 2281-2308. (14) Zhou, X.; Zhu, D.; Liao, Y.; Liu, W.; Liu, H.; Ma, Z.; Xing, D. Nat. protoc. 2014, 9, 1146-1159. (15) Miao, W. Chem. Rev. 2008, 108, 2506-2553. (16) Zhao, Z.; Zhou, X.; Xing, D. Biosens. Bioelectron. 2012, 31, 299-304. (17) Liu, W.; Zhou, X.; Xing, D. Biosens. Bioelectron. 2014, 58, 388-394. (18) Nishimasu, H.; Ran, F. A.; Hsu, P. D.; Konermann, S.; Shehata, S. I.; Dohmae, N.; Ishitani, R.; Zhang, F.; Nureki, O. Cell 2014, 156, 935-949. (19) Jiang, F.; Zhou, K.; Ma, L.; Gressel, S.; Doudna, J. A. Science 2015, 348, 1477-1481. (20) Wickramasinghe, S.; Fida, S. Blood 1994, 83, 1656-1661. (21) Spencer, J. P.; Whiteman, M.; Jenner, A.; Halliwell, B. Free Radical Bio. Med. 2000, 28, 1039-1050. (22) Kumar, S.; Cheng, X.; Klimasauskas, S.; Mi, S.; Posfai, J.; Roberts, R. J.; Wilson, G. G. Nucleic Acids Res. 1994, 22, 1.
5 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 6 of 6
For TOC only
6 ACS Paragon Plus Environment
