Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro

Nov 18, 2015 - (11-13) A wide spectrum of DNA-modifying enzymes, including DNA methyltransferases, restriction endonucleases, and DNA repair enzymes, ...
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Nucleosomes Inhibit Cas9 Endonuclease Activity in vitro John Michael Hinz, Marian F. Laughery, and John Jason Wyrick Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01108 • Publication Date (Web): 18 Nov 2015 Downloaded from http://pubs.acs.org on November 21, 2015

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Biochemistry

Nucleosomes Inhibit Cas9 Endonuclease Activity in vitro John M. Hinz, Marian F. Laughery, and John J. Wyrick* School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA 991647520 Supporting Information Placeholder ABSTRACT: During Cas9 genome editing in eukaryotic

cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospaceradjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins comprise an RNA-guided system for targeting viruses and other invading DNAs in some microbes.1 The CRISPR-Cas9 system is an increasingly popular tool for genome editing due to its ease in targeting site-specific DNA double strand breaks.1,2 In genome editing applications, targeting is typically controlled by a Cas9-bound single guide RNA (sgRNA).3,4 The 5' end of the sgRNA contains a 20-nucleotide guide sequence that is essential for DNA targeting, since it hybridizes to the genomic DNA target to generate a 20 bp RNA-DNA heteroduplex. In addition, a protospacer-adjacent motif (PAM) in the non-complementary DNA strand immediately adjacent to the RNA-DNA heteroduplex is required for Cas9 binding and endonuclease activity.3,5,6 The Streptococcus pyogenes Cas9 enzyme, which is commonly used for genome editing, cleaves guide RNA targets adjacent to a 5'-NGG-3' PAM motif. The mechanism of Cas9 binding and cleavage of DNA has been studied using naked DNA substrates.3,6,7 The current model is that Cas9 initiates DNA binding at PAM sites using a slow, three-dimensional diffusionbased search mechanism.7 Upon binding to DNA, Cas9 stimulates DNA unwinding, guide RNA hybridization, and ultimately DNA cleavage.7 During this process, the large Cas9 enzyme almost completely envelops at least

20 nucleotides of target DNA.8-10 Presumably, the DNA target must be fairly accessible for the Cas9 enzyme to initiate such an extensive binding interface. In genome editing applications, the bacterial Cas9 enzyme must target eukaryotic DNA that is packaged into chromatin. The primary subunit of chromatin is the nucleosome core particle, which is comprised of 147 bp of DNA wrapped 1.67 times around an octamer of histone proteins.11-13 A wide spectrum of DNA modifying enzymes, including DNA methyltransferases, restriction endonucleases, and DNA repair enzymes, have greatly reduced activity when their DNA substrate is present in a nucleosome.14-18 It is not known how Cas9 activity is affected by the packaging of its DNA substrate into nucleosomes. Anecdotal evidence indicates that Cas9 can cleave most target sequences within eukaryotic genomes to some degree regardless of the extent of chromatin packaging or accessibility. However, other evidence suggests that chromatin influences Cas9 binding in vivo. Chromatin immunoprecipitation-sequencing (ChIP-seq) studies have investigated the DNA binding distribution of a catalytically inactive dCas9 enzyme, and found that dCas9 binds many off-target sites that partially match the 20-nucleotide guide sequence.19,20 Importantly, offtarget dCas9 binding was found to strongly correlate with the degree of chromatin accessibility.19,20 These findings suggest that chromatin may regulate Cas9 binding and activity. In this study, we measured Cas9 activity on recombinant nucleosome substrates in vitro. We found that the Cas9 endonuclease is strongly inhibited when the PAM motif is located within a well-positioned nucleosome. In contrast, Cas9 activity is largely unaffected when the PAM motif is located within linker DNA, even if a large proportion of the target sequence is within the nucleosome core. These results suggest that the accessibility of the PAM motif is the critical determinant controlling Cas9 activity in chromatin. Cas9 Cleavage is inhibited on Nucleosome DNA. To investigate the effects of a nucleosome substrate on Cas9 endonuclease activity, we used an sgRNA (sgRNA1; see Figure S1A) to target the Cas9 endonuclease

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Figure 1. Cas9 cleavage inhibition in nucleosome core. A. Diagram of 289 bp DNA substrate showing the location of 601 nucleosome positioning sequence and the sgRNA target sites (white arrows, with PAM sequence at the arrowhead) and location of Cas9 nuclease cleavage (break in arrow). Arrow orientation reflects strand bound by the sgRNA, where right facing arrows denote sgRNA hybridization with the lower strand, and left facing arrows denote hybridization with the upper strand. B. Polyacrylamide gel electrophoresis showing cleavage of the DNA and nucleosome substrate by each Cas9/sgRNA complex after 1 hour at 37˚C. C. Chart of substrate cleavage after 1 hour for DNA and nucleosome substrates for each sgRNA. Columns represent the average of three to four independent experiments, and error bars represent standard deviations. Single asterisk denotes significant difference (P < 0.01) between the DNA and nucleosome substrates; double asterisk denotes P < 0.00001. to cleave a DNA substrate containing the 601 nucleosome positioning sequence.21 Since Cas9 acts as a single turnover enzyme under most in vitro reaction conditions,7 we determined the amount of Cas9 and sgRNA necessary to attain measurable product formation during a 1 hour incubation at 37°C with the 289 bp naked DNA substrate (Figure S1B). The targeted cut site for sgRNA1 is located within the 601 nucleosome positioning sequence (Figure S1A), and yielded the expected cleavage product of 170 bp (Figure S1B). Because the DNA substrate was radiolabeled on only one strand, the other 119 bp cleavage product was not detected in this assay. Using a range of Cas9/sgRNA1 concentrations, we determined that a 50:1 or greater ratio of Cas9/sgRNA1 to DNA substrate yielded almost complete cleavage of naked DNA (Figure S1D). We reconstituted nucleosomes using recombinant Xenopus laevis histones and the radiolabeled 289 bp DNA substrate (Figure S1C), and measured cleavage product formation on the resulting nucleosome substrate with increasing amounts of Cas9/sgRNA1 (Figure S1B, lower panel). There was a dose-dependent increase in the cleavage of the nucleosome substrate, but the overall cleavage activity was greatly reduced compared to that of naked DNA, with a maximum of ~10% cleavage product even at extremely high concentrations of the enzyme/sgRNA complex (Figure S1D). The reduction in activity is not simply due to the presence of histones in the reaction, as an equal amount of recombinant histone octamer was present in the naked DNA reactions to control for the possibility that the presence of soluble histones in the reaction impact Cas9 activity. A 50:1 ratio of Cas9/sgRNA to substrate was used as the reaction conditions for sub-

sequent experiments (see below). Taken together, these results suggest that Cas9 endonuclease activity is inhibited by a nucleosome substrate. Translational Position of Target Site Affects Cas9 Endonuclease Activity. To investigate how the translational position of the target site within the nucleosome affects Cas9 activity, we used sgRNAs that targeted different sites along the DNA substrate (Figure 1A). The sgRNA1 target site is between positions -26 to -48 relative to the nucleosome dyad (or pseudo-two-fold axis of symmetry; see Figure 1A). We designed a second guide RNA (sgRNA2) that targets within the nucleosome on the opposite strand, and on the opposite side of the dyad, at position +29 to +51. A third guide RNA (sgRNA3) targets a region that straddles the nucleosome core particle edge, extending between position +56 within the nucleosome and 6 bp into the linker DNA. A fourth guide RNA (sgRNA0) targets a region entirely within the linker DNA, 23 bp away from the nucleosome core boundary. While the choice of guide RNA targets was limited by the 601 sequence itself, the selected target sites span a range of translational positions within or adjacent to the 601 nucleosome. Cas9 activity with the different sgRNAs was generally high on naked DNA. The amount of cleavage product formation after 1 hour ranged from ~60% (sgRNA0) to as much as ~95% (sgRNA2) (Figure 1B, left panel). Presumably, this reflects small differences in sgRNA targeting efficiency, which is influenced by the nucleotide composition of the target DNA itself or sequences adjacent to the target site.22-24

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Figure 2. Accessibility of the PAM motif regulates Cas9 activity. A. Diagram of DNA substrate showing location of sgRNA3, sgRNA4, and sgRNA5 target sites (white arrows, with PAM sequence at the arrowhead) and location of Cas9 nuclease cleavage (break in arrow). See Figure 1A legend for more details. The sgRNA3 PAM is located outside the nucleosome core (PAM-out); the sgRNA4 PAM is inside the nucleosome core (PAM-in). B. Polyacrylamide gel analysis of the Cas9 cleavage products following 1 hour incubation with the DNA or nucleosome substrate. C. Quantification of substrate cleavage for each sgRNA. Columns represent the average of three to four independent experiments, and error bars represent standard deviations. Single asterisk denotes significant difference (P < 0.01) between the DNA and nucleosome substrates; double asterisk denotes P < 0.0001. In contrast, Cas9 activity on the nucleosome was strongly affected by the translational position of the target site relative to the nucleosome core position (Figure 1B, right panel). At sgRNA target sites located within the nucleosome, Cas9 activity was almost completed inhibited: sgRNA1 and sgRNA2 had ~85% and ~95% reduction in product formation on the nucleosome substrate relative to naked DNA (Figure 1C). In contrast, Cas9 cleavage of the sgRNA0 target, which falls in the linker DNA, was essentially unaffected by the presence of the adjacent nucleosome (P > 0.05). These data confirm that target sites within a positioned nucleosome core particle are poor substrates for Cas9 cleavage, but linker DNA can be readily cleaved. Notably, Cas9 activity at the sgRNA3 target site, which straddles the edge of the nucleosome core, showed an intermediate level of nucleosome inhibition. Cas9 activity at this site on the nucleosome substrate was significantly reduced compared to naked DNA (P < 0.05), but the magnitude of the inhibition was small (~19% reduction in product formation, see Figure 1C). These data suggest that Cas9 activity is only marginally inhibited when its target site is located at the edge of the nucleosome. Accessibility of the PAM is the Critical Determinant of Cas9 Activity on a Nucleosome. We wondered whether the high activity of Cas9 on the nucleosome at the sgRNA3 target site was because the PAM motif for this site was located outside the nucleosome boundary (PAM-out), in accessible linker DNA. To test this hypothesis, we designed an additional guide RNA (sgRNA4) to target the same location at the

nucleosome edge, but with the target site oppositely oriented so the PAM was located inside the nucleosome (PAM-in). If PAM accessibility is the critical determinant of Cas9 activity, then the sgRNA3 target site should be cleaved more efficiently than that of sgRNA4. However, if the accessibility of the target site as a whole is more important, than the sgRNA4 target site should be cleaved more efficiently, since it is shifted four nucleotides further into the linker DNA than sgRNA3 (Figure 2A). Cas9 activity at the sgRNA4 target site was significantly lower on the nucleosome substrate than naked DNA (Figure 2B-C). The relative decrease in Cas9 activity was comparable to that observed for sgRNA1 and sgRNA2 (Figure 1), which have target sites within the nucleosome interior, but less than the decrease observed at a target site located near the nucleosome dyad (sgRNA5; see Figure 2). Importantly, the degree of nucleosome inhibition was much greater at the sgRNA4 target site (~89% decrease in Cas9 activity) than sgRNA3 (~19% decrease). These results support the conclusion that PAM accessibility is the critical determinant of Cas9 activity in chromatin. It is known that Cas9 binding initiates at PAM motifs,7 and the sgRNA3 PAM site is located approximately 4-6 nucleotides beyond the nucleosome core boundary in accessible linker DNA. Cas9 binding to the sgRNA3 PAM site could initiate Cas9-catalyzed DNA unwinding that progressively invades the nucleosome, perhaps using a Brownian-ratchet mechanism.2527 In contrast, the sgRNA4 PAM site is located in the inaccessible nucleosome core, which could explain the strong inhibition of Cas9 cleavage at this site.

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In summary, our data show that nucleosomes inhibit Cas9 cleavage activity at target sites in which the PAM motif is located within the nucleosome core. As mononucleosomes comprise only the first level of chromatin packing, it is likely that higher-order chromatin structures within cells will be more inhibitory to Cas9 activity, unless Cas9 targeting stimulates local chromatin remodeling in vivo.28 It is known that guide RNA sequences can vary widely in their targeting efficiency and frequency of off-target effects.19,20,23,24 Our data indicate that chromatin is likely to significantly influence both of these critical parameters.

[8] Anders, C., Niewoehner, O., Duerst, A., and Jinek, M. (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease, Nature 513, 569-573. [9] Jinek, M., Jiang, F., Taylor, D. W., Sternberg, S. H., Kaya, E., Ma, E., Anders, C., Hauer, M., Zhou, K., Lin, S., Kaplan, M., Iavarone, A. T., Charpentier, E., Nogales, E., and Doudna, J. A. (2014) Structures of Cas9 endonucleases reveal RNA-mediated conformational activation, Science 343, 1247997. [10] Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., and Nureki, O. (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA, Cell 156, 935-949. [11] Davey, C. A., and Richmond, T. J. (2002) DNA-dependent divalent cation binding in the nucleosome core particle, Proceedings of

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[12] Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W., and Richmond, T. J. (2002) Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution, Journal of molecular biology 319, 1097-1113. [13] Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution, Nature 389, 251-260. [14] Polach, K. J., and Widom, J. (1999) Restriction enzymes as probes of nucleosome stability and dynamics, Methods in enzymology 304, 278-298. [15] Fatemi, M., Pao, M. M., Jeong, S., Gal-Yam, E. N., Egger, G., Weisenberger, D. J., and Jones, P. A. (2005) Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level, Nucleic acids research 33, e176. [16] Heo, K., Kim, H., Choi, S. H., Choi, J., Kim, K., Gu, J., Lieber, M. R., Yang, A. S., and An, W. (2008) FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16, Molecular cell 30, 86-97. [17] Trotter, K. W., and Archer, T. K. (2012) Assaying chromatin structure and remodeling by restriction enzyme accessibility, Methods in molecular biology 833, 89-102. [18] Rodriguez, Y., Hinz, J. M., and Smerdon, M. J. (2015) Accessing DNA damage in chromatin: Preparing the chromatin landscape for base excision repair, DNA repair. [19] Kuscu, C., Arslan, S., Singh, R., Thorpe, J., and Adli, M. (2014) Genomewide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease, Nature biotechnology 32, 677-683. [20] 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., Jaenisch, R., Zhang, F., and Sharp, P. A. (2014) Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells, Nature biotechnology 32, 670-676. [21] Fernandez, A. G., and Anderson, J. N. (2007) Nucleosome positioning determinants, Journal of molecular biology 371, 649-668. [22] Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B. L., Xavier, R. J., and Root, D. E. (2014) Rational design of highly active sgRNAs for CRISPR-Cas9mediated gene inactivation, Nature biotechnology 32, 1262-1267. [23] Wang, T., Wei, J. J., Sabatini, D. M., and Lander, E. S. (2014) Genetic screens in human cells using the CRISPR-Cas9 system, Science 343, 80-84. [24] Xu, H., Xiao, T., Chen, C. H., Li, W., Meyer, C. A., Wu, Q., Wu, D., Cong, L., Zhang, F., Liu, J. S., Brown, M., and Liu, X. S. (2015) Sequence determinants of improved CRISPR sgRNA design, Genome research. [25] Anderson, J. D., Thastrom, A., and Widom, J. (2002) Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation, Molecular and cellular biology 22, 7147-7157. [26] Astumian, R. D., and Bier, M. (1994) Fluctuation driven ratchets: Molecular motors, Physical review letters 72, 1766-1769. [27] Feynman, R. P., Leighton, R. B., and Sands, M. L. (1963) The Feynman lectures on physics, Addison-Wesley Pub. Co., Reading, Mass.,. [28] Polstein, L. R., Perez-Pinera, P., Kocak, D. D., Vockley, C. M., Bledsoe, P., Song, L., Safi, A., Crawford, G. E., Reddy, T. E., and Gersbach, C. A.

Supporting Information. Methods, supplemental Table S1, and supplemental Figure S1.

AUTHOR INFORMATION Corresponding Author

* Email: [email protected] Funding

This research was supported by National Institutes of Health (NIH) grants ES004106 (to Michael Smerdon, WSU) and ES002614 (J.J.W. and Michael Smerdon) from the National Institute of Environmental Health Sciences (NIEHS). Notes

The authors declare that they have no conflicts of interest with the contents of this article.

ACKNOWLEDGMENTS We thank Amelia Hodges, Dr. Peng Mao, and Dr. Michael Smerdon for helpful comments on the manuscript.

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the National Academy of Sciences of the United States of America 99, 11169-11174.

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Biochemistry (2015) Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators, Genome research 25, 1158-1169.

A. PAM in Nucleosome Core Cas9

B. PAM in Linker DNA Cas9

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