Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display

Abstract

Noncoding RNAs play diverse roles throughout biology and exhibit broad functional capacity. To investigate and harness these capabilities, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-Display (CRISP-Disp), a targeted localization method that uses Cas9 to deploy large RNA cargos to DNA loci. We demonstrate that functional RNA domains up to at least 4.8 kb long can be inserted in CRISPR guide RNA at multiple points, allowing the construction of Cas9 complexes with protein-binding cassettes, artificial aptamers, pools of random sequences and natural long noncoding RNAs. A unique feature of CRISP-Disp is the multiplexing of distinct functions at multiple targets, limited only by the availability of functional RNA motifs. We anticipate the use of CRISP-Disp for ectopically targeting functional RNAs and ribonucleoprotein (RNP) complexes to genomic loci.

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Figure 1: Large, structured RNA domains can be functionally appended onto the sgRNA scaffold at multiple points.
Figure 2: CRISPR-Display supports artificial and natural lncRNAs.
Figure 3: CRISPR-Display is compatible with structurally diverse RNA domains.
Figure 4: CRISPR-Display expands the functional repertoire of CRISPR-based methods.

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Acknowledgements

We thank L. Cong and F. Zhang for TALE plasmids and advice on reporter construct design; Y. Sancak, C. Fulco, S. Donovan and M. Morse for their general technical assistance; G. Kenty for his help with luminometry; D. Hendrickson and D. Tenen for their RIP expertise; C. Gerhardinger and C. Daly for their support with deep sequencing; and M. Tabebordbar, J. LaVecchio, S. Ionescu and M. Sauvageau for their assistance with FACS. We are grateful to all members of the Rinn laboratory for their thoughtful discussions and critiques. This work was supported by US National Institutes of Health grant P01GM099117.

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Authors

Contributions

D.M.S. designed and performed experiments; E.H. designed and performed microscopy experiments (Figs. 4c,d and Supplementary Figs. 13 and 14); S.T.Y. assisted with computational analysis (Fig. 3d and Supplementary Figs. 5 and 10); and J.L.R. directed research. D.M.S. and J.L.R. wrote the manuscript.

Corresponding author

Correspondence to John L Rinn.

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Competing interests

The authors declare competing financial interests. D.M.S. and J.L.R. have filed for patents concerning the design and expression of extended sgRNAs to expand the repertoire of dCas9 function.

Integrated supplementary information

Supplementary Figure 1 Project Overview, design and controls.

(a) Schematic of a general, targeted ncRNA localization system: a ncRNA cargo (and potential associated proteins) is ectopically targeted to a DNA locus via a programmable protein conduit. (b) Overview DNA targeting by S. pyogenes dCas9, directed by a minimal sgRNA that targets the GLuc reporter18. (c) Expression constructs in the dual reporter transcription activation system. Target and non-target sites in the Reporter and Normalizer are absent from the human genome Supplementary Table 1) and are multimerized to exploit the additivity of artificial transcription activators28,29. minCMV: a 60-nt minimal cytomegalovirus promoter; 2A: a 2A ‘self-cleaving’ peptide. Lentiviral variants are shown; in transient reporter assays, regions bracketed by asterisks are absent. CRISPR construct design is modeled after reference 18. (df) Reporter system visualization of locus-specific RNA-guided transcription activation in HEK293FT cells. (d) Live-cell fluorescence microscopy using transient reporters. All images are 10× magnification (e) FACS analysis of the same experiment. Cells were gated with an mCherry cotransfection control (data not shown). The percentages of Venus and mCerulean positive cells (mean, ± standard deviation, n = 3) are quoted. (f) Luciferase assays. Values are means ± standard deviation, n = 3.

Supplementary Figure 2 A proposed ncRNA ectopic localization system based on TALE two-hybrids.

(a) Top: System schematic and controls. Top, left: Direct activation by TALE~VP, a chimera of an optimized TALE domain30,39 and the VP64 transcription activator. Top, middle: Positive control for bridged activation in the TALE two-hybrid system. VP64 is replaced with the MS2 phage coat protein generating TALE~MS2. This is coexpressed with PP7~VP and a synthetic control RNA, derived from the T. thermophila Group I intron P4–P6 domain (Supplementary Fig. 4a), appended at its 3´ terminus with five copies of the MS2 stem-loop (5x MS2-SL), and internally with a cassette of five PP7 stem-loops (5x PP7-SL). The resulting ternary complex, TALE~MS2•RNA•PP7~VP, is termed the “5xSL Complex.” Top, right: Negative control for the TALE two-hybrid system. In the “0xSL Complex” the internal cassette of PP7 stem-loops is ablated. Bottom: Expression constructs used for this system. Reporter constructs are identical to those outlined in Supplementary Figure 1c. (b) FACS plots demonstrating direct activation by the TALE system, using integrated reporters. Top: baseline reporter expression in the presence of the unmodified TALE domain. Bottom: direct activation by TALE~VP. (c) Luciferase assays, as in Supplementary Figure 1f, using transient (left) and integrated (right) reporters. Values are means ± standard deviation; n = 3. (d) Components of the TALE system form the expected binary and ternary subcomplexes. Each control RNA (0xSL and 5xSL) was coexpressed with TALE~MS2 and PP7~VP, and immunoprecipitated with anti-FLAG (TALE) or anti-HA (PP7~VP) antibodies. Lane markers are shared for RIP (top) and western blot (bottom) panels. Inp, input. Top: RIP demonstrates the formation of TALE~MS2•[0xSL RNA], TALE~MS2•[5xSL RNA] and PP7•[5xSL RNA] subcomplexes. n/a, not applicable. Bottom: western blot on the same samples, demonstrating the formation of TALE~MS2•[5xSL RNA]•PP7~VP ternary complexes, but, as expected, not the corresponding complex with the 0xSL RNA. Inputs correspond to 2.5% of the starting sample; immunoprecipitates correspond to 12.5% of the recovered material. (e) Thermodynamic scheme summarizing the binding events within the TALE two-hybrid system, including the formation of binary complexes (2 and 3) and ternary complex (4) with the target DNA locus. “SL” refers to the 5xMS2-SL cassette. For clarity, reactions involving PP7 are omitted. When using integrated reporters, the concentration of target DNA is expected to be low (~1–2 copies/cell), far below those of TALE~MS2 and RNA~SL. When the concentration of TALE~MS2 furthermore exceeds that of RNA~SL, complex 3 is expected to saturate the target locus, outcompeting formation of complex 4.

Supplementary Figure 3 A split TALE approach to couple DNA-binding to RNA-binding in the TALE two-hybrid system.

(a) System schematic. The optimized TALE domain, which recognizes a 12-nt target30, is split into two smaller six-nt binding domains, N–TALE (fused to MS2) and C–TALE~VP (fused to PP7). Alone, these proteins should inefficiently recognize the full 12-nt target motif (bottom right). Coexpression of a “Splint RNA” bearing both cognate stem-loops should template assembly of a native-like TALE domain with higher competency (top right). (b) Cartoons summarizing splint RNAs tested. Each construct was under control of a U6 promoter; the splinting cassette (red and blue) preceded a 30-nt unstructured RNA common to all constructs. (c) Luciferase assays, demonstrating failure of the split TALE approach in its current design. Using transient reporters (top), the TALE domains induce reporter activation in the absence of a splint RNA. Coexpression of splint 1 reduces the apparent activity. n.d., no data. Using integrated reporters (bottom), no significant activation is seen with any of the splints. Values are means ± standard deviation; n = 3.

Supplementary Figure 4 Secondary structures of TOP1–4, INT, and Double TOP0–2 accessory domains; additional biochemical data.

(ad) Secondary structures of accessory domain components. (a) Secondary structure of the thermostable ΔC209 mutant of the T. thermophila group I intron P4–P6 domain, from which the accessory domains of TOP1–4, and their Double TOP analogs, were derived. Base pairing geometries are indicated using Leontis and Westhof nomenclature; the Watson-Crick G•A base pair introduced by the ΔC209 mutation is highlighted in blue. PP7-SL and MS2-SL cassettes (bd) were grafted into L6, boxed in gray. (b) Secondary structure of the 3xPP7 cassette used in U6-driven topology constructs. In TOP1–4, this cassette is inserted within P4–P6 stem P6b (top, left); the identical construct comprises the accessory domain of INT(P4–P6[3xPP7-SL]) (Fig. 3b,e,f). In INT, the cassette it is inserted within the sgRNA core (top, right). (c) Secondary structure of the 5xPP7 cassette used in Pol II–driven topology and double topology constructs (d) Secondary structure of the 5xMS2 cassette used in Double TOP0–2. (e,f) Additional biochemical data, in support of Figure 1. (e) FACS analyses on transient reporter assays using U6-driven TOP1, TOP2 and INT, as in Supplementary Figure 1e. Means ± standard deviation; n = 3. (e) RIP–qRT-PCR of dCas9•TOP1 and dCas9•TOP2. qPCR primers target the core sgRNA and the accessory domain (p1 and p2, respectively, right, Supplementary Table 3). Recovery of the sgRNA core and accessory domains from dCas9•TOP1 complexes was quantitative; from dCas9•TOP2 complexes the yield of accessory domain was nearly half that of the sgRNA core. Although this did not indicate a complete loss of the accessory domain, we hypothesize that other factors—such as RNA folding or the geometry of the dCas9•TOP2 complex—may contribute to the loss of TOP2-bridged activation. Furthermore, this result implies that structured 5´ additions might partially stabilize a modified sgRNA from degradation, as has been reported elsewhere20. Values are means ± standard deviation. n = 4. Student’s one-tailed t-test.

Supplementary Figure 5 CRISPR targeting specificity is not significantly altered by appending the sgRNA core with accessory RNA domains.

Global gene expression data, measured by poly(A)+ mRNA-Seq, from GLuc reporter HEK293FT cells transiently expressing dCas9~VP alone, or concomitantly with GLuc-targeting minimal sgRNA, TOP1, INT(3xPP7) or INT(P4–P6[3xPP7]) constructs (Supplementary Fig. 4). All data are averaged from two biological replicates. (a) Specific activation of the GLuc-Reporter by sgRNA and modified sgRNA constructs, observed by mRNA-seq, corroborates observations from luciferase and FACS assays. FPKM, fragments per kilobase million reads, as quantified with CuffDiff44. For comparison, values for the CLuc normalizer and dCas9~VP are shown. (b) Differential gene expression plots for the minimal and each modified sgRNA construct, relative to cells expressing dCas9~VP alone. All axes are on the same scale. The large blue circles correspond to the GLuc reporter locus; reads mapping to the (modified) sgRNAs–which were not observed in all experiments–have been masked from view. (c) Differential gene expression plots for each modified sgRNA, relative to the minimal sgRNA. In b and c, off-target genes with the highest fold changes in gene expression (B3GNT8, ELOVL5 and RPS17) are indicated; these were not measured as statistically significant. Moreover, no PAM-adjacent off-target binding sites complementary to the GLuc gRNA (defined by the 3´-most 10 nt of the guide) were observed within these loci and the adjoining 10 kb. From this, we hypothesize that sporadic activation of these genes may have resulted in response to expression of the modified sgRNA constructs, and not from guide-directed off-target activation by dCas9~VP.

Supplementary Figure 6 Surveying Pol II expression systems for CRISPR-Display.

(a) Schematics of RNA polymerase II (Pol II) expression systems for modified sgRNA constructs. Promoters and terminator elements are described in (Supplementary Note 2). For the CMV/MASC system, the RNase P cleavage point is indicated (red arrow). Modified sgRNA chimeras are as defined in Figure 1a and Supplementary Figure 4a,b. (b) Direct activation by Pol II–driven TOP constructs, measured via luciferase reporter assays. Values are means ± standard deviation. n = 3, Student’s one-tailed t-test, relative to negative controls (far left). “sg,” minimal sgRNA. (ce) Expression from the CMV/3´Box backbone appears to restore function to the TOP2 accessory domain. (c) Direct and bridged activation by the most effective constructs, using the most effective Pol II expression systems tested. Transient reporter assays are shown. Values are means ± standard deviation, n = 3; “sg,” minimal sgRNA, driven from a U6 promoter. (d) FACS analyses on transient reporter assays with CMV/3´Box constructs, as in Supplementary Figure 1e and 4e. Data for the sgRNA controls are the same as in Supplementary Figure 4e. Values are the means ± standard deviation. n = 3. (e) RIP/qRT–PCR of dCas9 complexed with CMV/3´-Box TOP1 or TOP2, as in Supplementary Fig. 4f. Positions of qPCR primer pairs are indicated in the schemas (left). Unlike U6-driven TOP2, under CMV/3´-Box expression, recovery of the sgRNA core and accessory domains was nearly quantitative. Values are means ± standard deviation, n = 4.

Supplementary Figure 7 The CMV/3´Box system generates non-polyadenylated, nuclear-localized transcripts.

(a) The majority of CMV/3´Box transcripts are nonpolyadenylated. Whole cell RNA was isolated from HEK293FT cells expressing TOP1–4 from CMV/SV40pA or CMV/3´Box backbones, and cDNA was synthesized in parallel reactions using random hexamer or oligo-dT primers. The apparent abundance of each construct was measured by qPCR using primer pair p2 (Supplementary Figs. 4f and 6e and Supplementary Table 3), and normalized to the signal observed with random hexamers. As a control, endogenous GAPDH was measured using the same protocol. (b,c) Subcellular fractionation quality controls. (b) Western blot analysis of four replicate samples. a/b-Tubulin and fibrillarin are cytoplasmic and nuclear markers, respectively. Whole cell lysates were generated from 5% of the initial samples, prior to fractionation, by boiling cells in RIPA buffer. 1 μg of protein was loaded onto each lane. (c) qRT-PCR analysis of three replicates. XIST and SNHG5 are nuclear and cytoplasmic ncRNAs, respectively. Data were processed as in RIP experiments, renormalizing observed Ct values to the total mass of RNA isolated from each subcellular compartment. Whole cell RNA was isolated from 5% of cells prior to fractionation. The percent yield, relative to whole cell RNA, reflects the abundance of transcript in each compartment. Values are means ± standard deviations; four technical replicates. Primers are listed in Supplementary Table 3. (d) CMV/3´Box transcripts are preferentially nuclear retained. Cells expressing TOP1 from each Pol II backbone were fractionated and analyzed as in c. The abundance in each compartment, relative to that observed with EF1α-SV40pA-TOP1-expressing cells, is shown. cDNA was primed with random hexamers; abundances were measured by qPCR using primer pair p2 (Supplementary Figs. 4f and 6e and Supplementary Table 3). Values are means ± standard deviations, n = 3.

Supplementary Figure 8 Integrated reporter luciferase assays with “Double TOP” constructs.

“Double TOP” constructs (defined as in Figure 2a and Supplementary Fig. 4) were expressed using the CMV/3´Box system; “sg,” minimal sgRNA, driven from a U6 promoter. Values are means ± standard deviations, n = 3. Significance was measured using Student’s one-tailed t-test, relative to negative control cells expressing dCas9~VP alone (far left).

Supplementary Figure 9 Synthesizing and sequencing the INT-N25 pool.

(a) Sequences and chromatograms of seven individual clones isolated during initial synthesis of the INT-N25 plasmid pool, aligned to the consensus (top). Nucleotides that match the consensus are notated as dots. Analysis and alignments were performed in Geneious (Biomatters Ltd). (b) Sequencing chromatogram of the aggregate INT-N25 pool, aligned to the consensus. Priming was initiated from a M15 reverse site located downstream of the sgRNA 3´ terminus (not shown). Heterogeneity 5´ of the insert is likely due to the small population of molecules containing inserts greater than 25 nucleotides in length (e.g., colony 5, above). (c) Schematic summarizing the primer design used to generate targeted deep sequencing libraries. Complete primer sequences are listed in Supplementary Table 6. (d) Bioanalyzer traces of the final sequencing libraries. For each, the expected length is 220 nt. Green and purple bands correspond to lane markers.

Supplementary Figure 10 Sequence diversity and expression of the INT-N25 pool.

(a) Read counts of 25-mers observed in the plasmid pool versus mean read counts observed in input (not immunoprecipitated) RNA libraries. Of the 783,612 unique sequences observed, 524 (0.07%) and 7,011 (0.9%) were significantly enriched or depleted in the input RNA libraries, respectively (red), as determined using DESeq2 (Ref. 48). Sequences containing more than five consecutive uridines, which act as Pol III termination signals, are among the depleted sequences. A zoomed view, representing the majority of data, is shown. (b) All data from the same experiment, plotted on a logarithmic scale.

Supplementary Figure 11 Design of the “Bunch of Baby Spinach” (BoBS) construct.

(a) Left: secondary structure of the 94-nt Spinach2 aptamer13, as observed in crystal structures. Right: predicted secondary structure of a minimal Spinach aptamer, “Baby Spinach.” A 29-nt double G-quadruplex core, which is responsible for binding DFHBI–1T, the fluorophore ligand13, is boxed in green. The core is abutted on both sides by base-paired stems, for which absolute sequence identity is thought to be inconsequential to activity. (b) Design of a 129-nt “BoBS” construct. Three tandem copies of the Baby Spinach core (green boxes) are embedded in a single, extended stem-loop, contiguous with the sgRNA core (gray box). (c) BoBS is brighter than Spinach2. INT-Spinach2 and INT-BoBS RNAs were prepared by in vitro transcription and folded by heat denaturation and buffer addition, as described13. Folded RNAs were serially diluted two-fold, incubated with 20 μM DFHBI-1T (Lucerna Technologies) and simultaneously imaged using a conventional UV trans-illuminator (Bio-Rad).

Supplementary Figure 12 Efficacy of INT-like CRISP-Disp constructs partially varies with length and expression level.

Direct activation activities (using integrated reporters, middle) and relative expression levels (bottom) for all INT-like constructs examined in Figure 3e, sorted according to insert length (top). Expression was quantified using RT-qPCR using universal primer pair pINT, which targets the sgRNA core outside of the INT insertion site (Supplementary Table 3). Values were normalized to internal GAPDH controls, and thereafter relative to the expression of the INT-S1 aptamer construct (far left); n = 4. Luciferase values are the same as plotted in Figure 3e. The most lowly expressed construct (INT-3xMS2 SL, expressed at 16% the level of INT-S1 Aptamer) exhibited activity within 90% of that of the INT-S1 aptamer construct. Conversely, the most highly expressed construct (INT-Spinach2, expressed at 160% the level of INT-S1 aptamer), exhibited 44% of the activity of the INT-S1 aptamer construct.

Supplementary Figure 13 Bridged imaging of genomic loci with CRISPR-Display.

(a) (Top) Experimental design. A telomere-targeting sgRNA18 internally appended with three MS2 stem-loops (“Telo-INT(3xMS2)”) binds an MS2~mCherry fusion, and is localized to the telomeric repeats by dCas9. (Bottom) Schematic of the MS2~mCherry expression construct. UBC: the human Ubiquitin Chain C promoter; MS2 (V75E/A81G) is a non-aggregating variant40; V5, a V5 epitope tag. dCas9 and modified sgRNA expression constructs are as defined in (Supplementary Fig. 1c). (b) Bridged telomere imaging requires a cognate sgRNA and dCas9. All cells express MS2~mCherry, in addition to the indicated constructs. “Telo-INT(3xK-T),” a telomere-targeting INT-like sgRNA appended with a cassette of three kink-turns (Supplementary Table 5). Images are merged z-stacks at 63× magnification, akin to Figure 4c. (c) Histogram of observed fluorescent puncta in 97 mCherry+ cells. Scale bar, 15 μm.

Supplementary Figure 14 Representative aptamer-based live cell images.

All cells are dCas9+, and express a telomere-targeting sgRNA18 internally appended with Spinach2 (Ref. 13), akin to the lower-right field in Figure 4d. Images are merged z-stacks at 63× magnification. Scale bar, 15 μm.

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Shechner, D., Hacisuleyman, E., Younger, S. et al. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat Methods 12, 664–670 (2015). https://doi.org/10.1038/nmeth.3433

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