11 A Protocol for Non-biased Identification of RNAs Transferred
Between Heterologous Mammalian Cell Types Using RNA Tagging,
Cell Sorting, and Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Sandipan Dasgupta and Jeffrey E. Gerst
12 Synthesizing Fluorescently Labeled dsRNAs and sRNAs
to Visualize Fungal RNA Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Rachael Hamby, Ming Wang, Lulu Qiao, and Hailing Jin
13 Labeling of dsRNA for Fungal Uptake Detection Analysis . . . . . . . . . . . . . . . . . . . 227
Matteo Galli, Jafargholi Imani, and Karl-Heinz Kogel
PART IV IDENTIFICATION AND ANALYSIS OF RNA-BINDING PROTEINS
14 Using RNA Affinity Purification Followed by Mass Spectrometry
to Identify RNA-Binding Proteins (RBPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Mengge Shan and Brian D. Gregory
15 Plant Individual Nucleotide Resolution Cross-Linking
and Immunoprecipitation to Characterize RNA-Protein Complexes . . . . . . . . . . 255
Tino Ko ¨ster and Dorothee Staiger
16 A Single-Molecule RNA Mobility Assay to Identify Proteins
that Link RNAs to Molecular Motors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Varun Bhaskar, Min Jia, and Jeffrey A. Chao
17 Proximity-CLIP Provides a Snapshot of Protein-Occupied RNA
Elements at Subcellular Resolution and Transcriptome-Wide Scale. . . . . . . . . . . . 283
Daniel Benhalevy and Markus Hafner
18 Double-Stranded RNA Pull-Down to Characterize Viral Replication
Complexes in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Marco Incarbone and Christophe Ritzenthaler
PART V RNAS AS GUIDES FOR GENOME EDITING AND IMAGING
OF CHROMOSOME LOCI
19 CRISPR Guide RNA Design Guidelines for Efficient Genome Editing . . . . . . . . 331
Patrick Schindele, Felix Wolter, and Holger Puchta
20 Live-Cell CRISPR Imaging in Plant Cells with a Telomere-Specific
Guide RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Solmaz Khosravi, Steven Dreissig, Patrick Schindele, Felix Wolter,
Twan Rutten, Holger Puchta, and Andreas Houben
21 Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular
Beacon Hybrid Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Xiaotian Wu, Yachen Ying, Shiqi Mao, Christopher J. Krueger,
and Antony K. Chen
22 Using RNA Tags for Multicolor Live Imaging of Chromatin Loci
and Transcription in Drosophila Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Hongtao Chen and Thomas Gregor
viii
Contents
Between Heterologous Mammalian Cell Types Using RNA Tagging,
Cell Sorting, and Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Sandipan Dasgupta and Jeffrey E. Gerst
12 Synthesizing Fluorescently Labeled dsRNAs and sRNAs
to Visualize Fungal RNA Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Rachael Hamby, Ming Wang, Lulu Qiao, and Hailing Jin
13 Labeling of dsRNA for Fungal Uptake Detection Analysis . . . . . . . . . . . . . . . . . . . 227
Matteo Galli, Jafargholi Imani, and Karl-Heinz Kogel
PART IV IDENTIFICATION AND ANALYSIS OF RNA-BINDING PROTEINS
14 Using RNA Affinity Purification Followed by Mass Spectrometry
to Identify RNA-Binding Proteins (RBPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Mengge Shan and Brian D. Gregory
15 Plant Individual Nucleotide Resolution Cross-Linking
and Immunoprecipitation to Characterize RNA-Protein Complexes . . . . . . . . . . 255
Tino Ko ¨ster and Dorothee Staiger
16 A Single-Molecule RNA Mobility Assay to Identify Proteins
that Link RNAs to Molecular Motors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Varun Bhaskar, Min Jia, and Jeffrey A. Chao
17 Proximity-CLIP Provides a Snapshot of Protein-Occupied RNA
Elements at Subcellular Resolution and Transcriptome-Wide Scale. . . . . . . . . . . . 283
Daniel Benhalevy and Markus Hafner
18 Double-Stranded RNA Pull-Down to Characterize Viral Replication
Complexes in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Marco Incarbone and Christophe Ritzenthaler
PART V RNAS AS GUIDES FOR GENOME EDITING AND IMAGING
OF CHROMOSOME LOCI
19 CRISPR Guide RNA Design Guidelines for Efficient Genome Editing . . . . . . . . 331
Patrick Schindele, Felix Wolter, and Holger Puchta
20 Live-Cell CRISPR Imaging in Plant Cells with a Telomere-Specific
Guide RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Solmaz Khosravi, Steven Dreissig, Patrick Schindele, Felix Wolter,
Twan Rutten, Holger Puchta, and Andreas Houben
21 Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular
Beacon Hybrid Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Xiaotian Wu, Yachen Ying, Shiqi Mao, Christopher J. Krueger,
and Antony K. Chen
22 Using RNA Tags for Multicolor Live Imaging of Chromatin Loci
and Transcription in Drosophila Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Hongtao Chen and Thomas Gregor
viii
Contents