1. Introduction
2. Materials and Methods
2.1. HEK293 Cell Culture
2.2. dCas9 and Guide Cloning and Guide Design
2.3. Stable Cell Line Production
2.4. CAPTURE and Proteomics
2.5. Western Blotting
2.6. Chromatin Immunoprecipitation (ChIP)
2.7. ChIP-Seq
2.8. Bioinformatics Analysis
2.9. ChIP-Seq Data Processing
2.10. Chromatin RNA-Seq Data Processing
3. Results
3.1. Designing Guides for DCas9 Targeting
3.2. Testing the CAPTURE System
3.3. CAPTURE on the U2 snRNA Gene PSE and DSE
3.4. SPT6 Helps to Recruit Integrator to the Human snRNA Genes
3.5. CDK12 Is Associated with the U2 snRNA Genes
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Forward Primer | Reverse Primer |
---|---|---|
dCas9 EcoVR | GAATGATATCGTGCTGACCCTGACAC | GTCCACATCGTAGTCGGACAG |
dCas9 FseI | GCCATCGTGCCTCAGAGCTTTC | GAACGGCCGGCCTTTTTCGTGG |
dCas9 ExF Seq | GGGCACATACCACGATCTGCTG | |
dCas9 Mut Seq | GGCAAGAGCGACAACGTGCCCTC | |
dCas9 ExR Seq | GGATTCTCCTCGACGTCACCGC | |
G1 | CACCCCGCCCTTTCACAGAGGGCG | AAACCGCCCTCTGTGAAAGGGCGG |
G2 | CACCATGCCCCGCCCTTTCACAGA | AAACTCTGTGAAAGGGCGGGGCAT |
G3 | CACCAAATGAAAGCCCGGGAACGC | AAACGCGTTCCCGGGCTTTCATTT |
G4 | CACCTCTCATCCACATTCAAGTCG | CACCTCTCATCCACATTCAAGTCG |
G5 | CACCGACGGTGACGGCGGGCGCGA | AAACTCGCGCCCGCCGTCACCGTC |
G6 | CACCAAGGCGAGCGCATCGCTTCT | CACCAAGGCGAGCGCATCGCTTCT |
G7 | CACCCCGTCTACCGCCCGCACATC | AAACGATGTGCGGGCGGTAGACGG |
G8 | CACCGCACGCTGTCGTTTCCACCG | CACCGCACGCTGTCGTTTCCACCG |
G9 | CACCCCGGAAGGGTTTCGCGTCAT | CACCCCGGAAGGGTTTCGCGTCAT |
G10 | CACCAGTGGGTGGCGACCTTTTAA | CACCAGTGGGTGGCGACCTTTTAA |
U2 N | GGAGCGGAGCGTTCTCTGTCTCCCC | AGAGTGTGAGCCCTCATTCACGCCC |
U2 DSE | TGGCTCGATACGAACAAGGAAG | GTTCCCGGGCTTTCATTTCG |
U2 UpPSE | GGGAACGCCGAAGAAGCACGGG | CCCCAGCCTCGCTCCTTGCCC |
U2 PSE | ATGAGAGTGGGACGGTGA | CACTTGATCTTAGCCAAAAGG |
U2 3′ box | ACGAGTCCTGTGACGCGCCGGCTTG | CTCCGGGTGGGTCCCATTCCTTTAA |
U2 CT-rich | CCTCCCCGCCTCTCCCTCGCTC | GGACAAATAGCCAACGCATGCGG |
U1 PSE | GGAAAGGGCTCGGGAGTGCGCG | CAGGTAAGTATGAGAGCTTGGGC |
Chr6 off target | AGACTACACGATACAACATCCAC | AGCAGGAATCAGAACTCCCATC |
Chr7 off target | AGCAGGAATCAGAACTCCCATC | ACCAAGGAGGAAAGGTAGTAGC |
Antibodies | Reference |
---|---|
AffiniPure Donkey Anti-Rabbit IgG (H + L) | Jackson ImmunoResearch 711-005-152 |
Anti-CRISPR-Cas9 | Abcam ab191468 |
CDK12 | LS Bio LS-C288466-100 |
CDK12 | Novus Biologicals NB100-87011 |
Cyclin K | Bethyl Laboratories A301-939A |
Oct-1 | Bethyl Laboratories A301-717A |
RNA Polymerase II | Novus Biologicals NBP2-32080 |
SPT6 | Cell Signalling 15616 |
Streptavidin (HRP) | Abcam ab7403 |
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