We have developed CRISPR-assisted RNA–protein interaction detection method (CARPID), which leverages CRISPR–CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific long non-coding RNAs (lncRNAs) in the native cellular context. We applied CARPID to the nuclear lncRNA XIST, and it captured a list of known interacting proteins and multiple previously uncharacterized binding proteins. We generalized CARPID to explore binders of the lncRNAs DANCR and MALAT1, revealing the method’s wide applicability in identifying RNA-binding proteins.
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Sequencing data have been deposited in the GEO under the accession number GSE137556. The secondary structure prediction data of XIST lncRNA from PARIS analysis used in Extended Data Fig. 2 were from a previous study (GEO accession: GSE74353)10 and are available in Supplementary Data 1 after additional processing. The raw sequence reads for TAF15 HTR-SELEX are available in Supplementary Data 2. Mass spectrometry raw data have been deposited to iProX database under the project ID number IPX0001797000. The TAF15 CLIP-seq data used in Extended Data Fig. 4c,d were from a previous study (GEO accession: GSE77700)12. Source data are provided with this paper.
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We are grateful to B. Ren (UC San Diego) and D. Leung (HKUST) for insightful comments during manuscript preparation. This work was supported by the City University of Hong Kong (7200595, 7005314, 9667188 and 9610424 to J. Yan), the National Natural Science Foundation of China (81873642 to J. Yan and 31900443 to W.S.), Research Grants Council of Hong Kong (21100615, 11102118, 11101919, C7007-17GF to K.M.C., and 21101917, 11103318 to L.Z.), the Shenzhen Science and Technology Fund Program (JCYJ20170818104203065, JCYJ20180307124019360 to K.M.C., JCYJ20170413141047772, JCYJ20180507181659781 to L.Z.); Opening Foundation of Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), the Chinese Ministry of Education (J. Ye); and the Hong Kong Epigenomics Project of the EpiHK consortium (Lo Ka Chung Charitable Foundation) (J. Yan and K.M.C.).
The authors declare no competing interests.
Peer review information Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, The location of the three sets of gRNAs on XIST. b, Scheme of BASU-dCasRx construct. c, Test of BASU efficiency at different length of reaction time in HEK293T cells. Three independent experiments were carried out with similar results and a representative result is shown. d, Scatter plots show comparison of gene expression level in HEK293T cells (WT) or cells transfected with BASU-dCasRx plus different gRNA sets (XIST-L1/XIST-L2/XIST-L3). The axes in each figure represent the log2 transformed gene expression. The expression level of XIST gene in each figure is highlighted (n = 3 independent experiments, two-sided Pearson correlation test).
a, RT-qPCR shows the specificity of the CRISPR/CasRx system in XIST normalized with GADPH. Data are represented as mean ± SEM, n = 3 independent experiments, one-sided ANOVA. b, The genome browser view of XIST-L1/L2/L3 over the XIST hairpin structure information resulted from Lu et al.10, using black strings to represent the region with complementary pairing. Vertical lines in different colors highlight the locations of different gRNA sets targeting loci on XIST lncRNA. The locations of known structural domains (A-H) are also indicated.
a, The Venn diagram illustrates the overlap of MS-detected proteins with ≥ 2 peptides in CARPID assays among the three different sets of gRNAs (XIST-L1/XIST-L2/XIST-L3). b, The Venn diagrams illustrate the overlap of MS-detected proteins with ≥ 2 peptides among the three independent experiments using the same gRNA set (Replicate#1/Replicate#2/Replicate#3). c, The Venn diagram illustrates the overlap of significantly enriched RBPs of XIST identified by CARPID using different sets of gRNAs (XIST-L1/XIST-L2/XIST-L3). d, The volcano plots show the enrichment of XIST-associated proteins in HEK293T cells. Significantly enriched proteins are labelled as orange dots. Proteins previously known to interact with XIST are labelled in orange font. SNF2L and TAF15, two novel XIST-associated proteins, are in blue font (n = 3 independent experiments for control and each gRNA group, respectively).
a, White nodes indicate proteins identified specifically by one set of gRNAs. Pink nodes represent proteins identified by two sets of gRNAs. Red nodes show proteins identified by all three sets of gRNAs. Edges are drawn between two proteins with annotated interaction (STRING interaction score ≥ 0.40). The width of the edge is adjusted in proportion to the STRING interaction score. Nodes with purple edges highlight the proteins involved in chromatin remodeling. Previously reported XIST-associated proteins are highlighted in bold orange font. SNF2L and TAF15 are indicated by blue font. b, The top five significant GO terms for the XIST-associated proteins (n = 35 genes were included, Fisher’s exact test). c, Genome browser shows the binding of TAF15 to XIST using CLIP-seq data from mouse brain tissues12. Non-redundant reads are separately shown for both strands. d, Bar-plot shows the number of TAF15 peaks on XIST compared to the expected peak number (mean ± SD). The peak loci are from Ref. 12. One-sided Poisson test (p-value).
a, The scheme of oligo design for HTR-SELEX. These oligos contain T7 promoter, Illumina adaptor (P5/P7), and 40-nt random sequence. b, Schematic representation of the HTR-SELEX experiment. c, RNA binding motif of TAF15 enriched from HTR-SELEX analysis. d, The scheme of machine learning.
a, Top: Western blot detection of SNF2L in input and streptavidin IP samples of control (Control) and three XIST gRNA sets (XIST-L1, XIST-L2 and XIST-L3). Bottom: immunoFISH images of XIST and SNF2L in HEK293T cells. The white boxed region on the left is magnified and shown on the right. Three independent experiments were carried out with similar results and a representative result is shown. b, Validation of XIST-SNF2L interaction using formaldehyde-assisted RIP assay (mean ± SEM, n = 3 independent experiments, two-sided paired Student’s t-test).
a, Representative microscopy images of GFP. Conditions are the same as in Fig. 1f. Three independent experiments were carried out and a representative result is shown. b, Validation of shRNA knockdown efficiency. β-actin was used to normalize the RNA expression (mean ± SD, n = 3 independent experiments, one-sided ANOVA test). c, Rescue efficiency of TAF15 knockdown. TAF15 expression was measured with RT-qPCR, and β-actin was used as an internal control (mean ± SD, n = 3 independent experiments, two-sided unpaired Student’s t-test). d, GFP expression upon TAF15 knockdown and rescue experiments. GFP expression under the same experimental conditions as in panel c was determined by RT-qPCR (mean ± SD, n = 3 independent experiments, two-sided unpaired Student’s t-test). e, Autosomal genes transcription under depletion of TAF15 in iMEF. Knockdown of TAF15 were conducted using two different shRNAs in female iMEF cells (E2C4) the same as in Fig. 1f. The expression levels were determined by RT-qPCR and normalized to β-actin (mean ± SEM, n = 3 independent experiments, one-sided ANOVA test with NT+5-aza as controls). f, Allelic RNA-seq confirms the role of TAF15 on XCI. Genes exhibiting significant allelic expression changes upon 5-aza treatment (NT+5-aza vs. NT) are grouped into X-chromosomal (chrX) genes and autosomal genes. By scaling the expression ratio between minor and major alleles before treatment as 0 (unbalanced) and after treatment as 1 (balanced), the allelic ratio of Taf15 knockdown is summarized separately for X-chromosomal (blue) and autosomal genes (grey). Data are represented as mean ± SEM, two independent experiments, two-sided unpaired Student’s t-test. g, A working model for the dual role of XIST lncRNA in mediating XCI.
a, Scatter plots show comparison of gene expression level in HEK293T cells (WT) or cells transfected with pre-gRNA sets (control). The axes in each figure represent the log2 transformed gene expression. The expression levels of XIST/MALAT1/DANCR are highlighted (n = 3 independent experiments, two-sided Pearson correlation test). b, The location of gRNA sets (Upper: DANCR-L1/2; Lower: MALAT1-L1/L2) and qPCR primers (Upper: DANCR: P1/P2; Lower: MALAT1: P1/P2) for DANCR used in CARPID. F, forward strand primer; R, reverse strand primer. The numbers within parentheses indicate the location of the gRNA sets in the corresponding RNA transcript, starting from 1 nt. c, The volcano plots show the enrichment of DANCR-associated proteins in HEK293T cells. Significantly enriched proteins are labelled as orange dots (n = 3 independent experiments for control and each gRNA group, respectively). d, DANCR-protein interaction network. White nodes indicate proteins identified with one set of gRNAs. Pink nodes represent proteins identified with two sets of gRNAs. Edges are drawn between two proteins with annotated interaction (STRING interaction score ≥ 0.40). The width of the edge is adjusted in proportion to the STRING interaction score.
a, Schematic representation of exosome isolation from cultured human cells. Exosome used for DANCR detection is highlighted in red font. b, Immunoblotting to examine the purification of exosome. 5 µg or 10 µg of cell lysates and exosomes fractions were resolved on the SDS-PAGE gel and subjected to Western blots of the indicated proteins. Note that CD81 was highly enriched in purified exosomes. Three independent experiments were carried out with similar results and a representative result is shown. c, Comparison of levels of XIST in HEK293T whole cell lysates and exosomes. XIST: P1/P2 represent two different sets of qPCR primers for XIST detection (as indicated in Extended Data Fig. 1a). Data are represented as mean ± SD, n = 3 independent experiments with three technical replicates. p values are calculated using two-sided unpaired Student’s t-test.
a, MALAT1-protein interaction network. White nodes indicate proteins identified with one set of gRNAs. Pink nodes represent proteins identified with two sets of gRNAs. Edges are drawn between two proteins with annotated interaction (STRING interaction score ≥ 0.40). The width of the edge is adjusted in proportion to the STRING interaction score. b, The volcano plots show the enrichment of MALAT1-associated proteins in HEK293T cells. Significantly enriched proteins are labelled as orange dots (n = 3 independent experiments for control and each gRNA group, respectively). c, Comparison of CARPID results among different lncRNAs.
Full blots for western blot in Fig. 1.
Full blots for western blot in Fig. 2.
Full blots for western blot in Extended Data Fig. 1.
Full blots for western blot in Extended Data Fig. 6.
Full blots for western blot in Extended Data Fig. 9.
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Yi, W., Li, J., Zhu, X. et al. CRISPR-assisted detection of RNA–protein interactions in living cells. Nat Methods 17, 685–688 (2020). https://doi.org/10.1038/s41592-020-0866-0
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