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Genomic Targeting of Epigenetic Probes Using a Chemically Tailored Cas9 System

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Genomic Targeting of Epigenetic Probes Using a Chemically Tailored Cas9 System Genomic targeting of epigenetic probes using a chemically tailored Cas9 system Glen P. Liszczaka, Zachary Z. Browna, Samuel H. Kima, Rob C. Oslunda, Yael Davida, and Tom W. Muira,1 aDepartment of Chemistry, Princeton University, Princeton, NJ 08544 Edited by James A. Wells, University of California, San Francisco, CA, and approved December 13, 2016 (received for review September 20, 2016) Recent advances in the field of programmable DNA-binding proteins Here, we report a method that combines the versatility of have led to the development of facile methods for genomic pharmacologic manipulation with the specificity of a genetically localization of genetically encodable entities. Despite the extensive programmable DNA-binding protein. Our strategy uses a utility of these tools, locus-specific delivery of synthetic molecules chemically tailored dCas9 to display a pharmacologic agent at a remains limited by a lack of adequate technologies. Here we combine genetic locus of interest in live mammalian cells (Fig. 1). This is trans the flexibility of chemical synthesis with the specificity of a pro- accomplished using split intein-mediated protein -splicing grammable DNA-binding protein by using protein trans-splicing to (PTS) to site-specifically link a recombinant dCas9:guide RNA ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) (gRNA) complex to the synthetic cargo of choice (22). Indeed, in vitro and subsequently deliver the dCas9 cargo to live cells. we show that the remarkable specificity, efficiency, and speed of PTS allow the direct generation of desired dCas9 conjugates The versatility of this technology is demonstrated by delivering within the cell culture media, thereby facilitating a streamlined dCas9 fusions that include either the small-molecule bromodomain “one-pot” approach for genomic targeting of the reaction product. and extra-terminal family bromodomain inhibitor JQ1 or a pep- We used this strategy to direct the epigenetic probes JQ1 (a tide-based PRC1 chromodomain ligand, which are capable of small molecule) and UNC3866 (a modified peptide) to spe- recruiting endogenous copies of their cognate binding partners cific genomic loci, where they interact with their target his- to targeted genomic binding sites. We expect that this technology tone recognition domains, the bromodomain and extra-terminal will allow for the genomic localization of a wide array of small (BET) family bromodomains and Polycomb repressive complex molecules and modified proteinaceous materials. 1 (PRC1) chromodomains, respectively. Therefore, we offer a strategy to direct endogenous epigenetic proteins and macro- chemical biology | epigenome engineering | protein delivery | protein molecular complexes to specific loci in the absence of genetic semisynthesis | intein splicing manipulation or overexpression of exogenous protein. Further- more, the highly modular nature of this approach makes it easily ll eukaryotic genomes are exquisitely packaged into the adaptable to a wide variety of synthetic entities. Anucleus as chromatin, wherein DNA is wrapped around Results histone proteins. To achieve the transcriptional output associ- Loci-Specific Delivery of Modified dCas9. Several bioconjugation ated with a particular cell type, chromatin is dynamically deco- techniques, including maleimide chemistry and HaloTag label- rated with a variety of covalent modifications and effector ing, have been used previously to conjugate synthetic moieties, molecules at specific genetic loci (1, 2). The resulting epigenomic such as biotin and fluorophores, to Cas9 (23, 24). PTS offers landscape establishes proper DNA accessibility and genomic struc- several advantages over these methods that make it ideal for our ture (3, 4), and its misregulation can serve as an underlying cause of approach; importantly, reactions can be performed in cell media, genetic disorders, including cancer (5). Thus, deliberate perturba- are complete within minutes, and do not require downstream tion of chromatin effector function has become a cornerstone for purification, thus allowing us to directly deliver the reaction dissecting the molecular mechanisms of transcription regulation pathways in live cells. Significance The activity of chromatin regulatory proteins can be manipu- lated using both genetic and pharmacologic approaches (6, 7). Programmable DNA-binding proteins such as nuclease-deficient The former includes technologies that alter the total cellular Cas9 (dCas9) offer a simple system for genomic localization of levels of a target transcript through transgene overexpression, genetically encodable biomolecules. These tools have enabled knockdown, or knockout, whereas the latter encompasses the use characterization of numerous different chromatin effectors at of small-molecule chemical probes that can modulate the func- specific genetic elements. The delivery of fully synthetic cargos tional output of a protein of interest. Although these techniques to specific loci is currently limited by a lack of adequate tech- are capable of globally affecting protein activities, locus-specific nologies. Here we used protein trans-splicing to ligate chemical epigenomic manipulation has been demonstrated to be a more moieties to dCas9 in vitro and subsequently deliver these mol- effective method for probing downstream transcriptional outputs ecules to live cells. We demonstrate the effectiveness and ver- – (8 10). Targeted epigenome engineering is accomplished by fusing satility of this method by delivering both small-molecule and genetically encodable chromatin effector proteins to programmable proteinaceous epigenetic probes to endogenous genomic sites DNA-binding proteins, such as transcription activator-like effectors where they interact with their target proteins. This facile and (11), zinc fingers (12), and nuclease-deficient Cas9 (dCas9) modular strategy will find broad use in locus-specific targeting – (13 16). A complementary approach to the genetic fusion strategy of chemical cargos to study nuclear processes. is genomic localization of purely synthetic entities. This opens the door for subnuclear targeting of epigenetic probes, including Author contributions: G.P.L. and T.W.M. designed research; G.P.L., Z.Z.B., S.H.K., R.C.O., small molecules and modified proteinaceous material (17, 18). and Y.D. performed research; G.P.L. and T.W.M. analyzed data; and G.P.L. and T.W.M. Examples of synthetic targeting agents include pyrrole-imidazole wrote the paper. polyamides and peptide nucleic acids (19, 20). Despite the ele- The authors declare no conflict of interest. gance of these strategies, however, their widespread application This article is a PNAS Direct Submission. has been limited by the need for specialized chemical synthesis 1To whom correspondence should be addressed. Email: [email protected]. methods and by constraints associated with their genomic tar- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BIOCHEMISTRY geting (i.e., locus specificity) (19, 21). 1073/pnas.1615723114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1615723114 PNAS | January 24, 2017 | vol. 114 | no. 4 | 681–686 Downloaded by guest on September 30, 2021 purification, i.e., a one-pot process. We found that the splicing reaction was >90% complete within 20 min, at which point we used a previously reported cationic lipid-mediated delivery strategy (25) to introduce the modified dCas9-gRNA complexes at an optimal concentration of 100 nM into 293T cells (SI Ap- pendix, Fig. S3). Delivery of the dCas9-VP64:gRNA complexes into cells led to transcriptional activation of the gRNA target gene, as measured by reverse-transcription quantitative PCR (RT-qPCR) analysis (Fig. 2C). Moreover, the level of mRNA induction was similar to activation levels observed following delivery of the recombinantly expressed dCas9-VP64 genetic fusion protein counterpart (SI Appendix,Fig.S4), and the signal was completely Fig. 1. Strategy for localizing synthetic molecules to specific genomic loci using a modified CRISPR/Cas9 system. Purified dCas9-IntN, IntC-cargo, and gRNAs are mixed in a one-pot reaction (1) to afford a dCas9-cargo:gRNA complex along with the excised intein fragments. This reaction mixture is then directly delivered to live cultured cells using a cationic lipid-mediated transfection strategy (2). Once inside the cell, the dCas9-cargo:gRNA com- plex enters the nucleus and binds to its target DNA sequence, resulting in localized activity of the synthetic cargo (3). product to cells. In addition, the intein tags are excised from the final construct and thus will not effect Cas9 delivery or down- stream applications. To explore the feasibility of our proposed methodology, we used a PTS reaction to ligate dCas9 to a potent transcription- Fig. 2. Cellular delivery of PTS-modified dCas9. (A) Schematic of the splicing activating peptide, VP64 (Fig. 2A). Previous studies have shown reaction between recombinantly expressed dCas9-NLS-IntN (1) and IntC- that when targeted to the promoter region of genes such as VP64-HA yields the dCas9-NLS-VP64-HA delivery product (2) and the inert interleukin-1 receptor antagonist (IL1RN) and neurotrophin-3 split intein complex. (B) The reaction from A as monitored by an SDS/PAGE NTF3 gel shift assay. The dCas9-NLS-IntN starting material (1) undergoes conver- ( ), the dCas9-VP64 fusion protein recruits transcriptional sion to the dCas9-NLS-VP64 delivery product (2) (96% in 20 min as measured machinery that induces local histone modifications,
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