A Comparison of Two Versions of the CRISPR-Sirius System for the Live-Cell Visualization of the Borders of Topologically Associating Domains
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmid Construction
2.2. Cell Culture and Transient Transfection
2.3. Lentivirus Production and Cell Transduction
2.4. FACS
2.5. Western Blot
2.6. Real-Time PCR
2.7. Live-Cell Microscopy
2.8. Graphical Representation of Hi-C and ChIP-Seq Data and Selection of Appropriate TAD Boundaries for Visualization
2.9. Epigenetic Data
3. Results
3.1. Evaluation of the PCP Version of the CRISPR-Sirius System
3.2. Evaluation of the MCP Version of the CRISPR-Sirius System
3.3. Evaluation of the Imaging Performance Using a Single Guide RNA per Locus
3.4. Analysis of the Expression of sgRNAs and Stem-Loop-Binding Proteins in Two Versions of the CRISPR-Sirius System
3.5. Expanding the Set of Target Loci—Visualizing the Boundaries of TADs
3.6. Analysis of the Dependence of the Visualization Efficiency on the Number of sgRNA Repeats in a Cluster and Epigenetic Factors
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Primer Sequence | Task |
---|---|---|
T2A_BamH_f | AATAAGGATCCGAGGGCAGAGGAAGTCTTCTAACAT | Replacing the P2A-HSA fragment in the dCas9 vector with the T2A-Puro fragment |
Puro_Xba_r | AATAATCTAGATAGATCAGGCACCGGGCTT | |
sfGFP_BamH_f | ATTAAGGATCCATGCGTAAAGGCGAAGAGCT | Replacing the HaloTag with sfGFP in a plasmid with the MCP gene |
sfGFP_Xho_r | TAATTCTCGAGTTTGTACAGTTCATCCATACCATGCG |
Target Locus 1 | sgRNA Recognition Sequence |
---|---|
IDR3 (chr19: 380,836–382,654) | AGCAGATGTAGG (45) 2 |
C6 (chr6: 157,310,367–157,314,361) | sg1: GTGAGTGCACAC (22) sg2: TGGGACACTATGATG (39) |
Target | PCP-sfGFP Version | MCP-sfGFP Version | ||
---|---|---|---|---|
Transient Transfection | Stable Transduction | Transient Transfection | Stable Transduction | |
IDR3 | 6% (n = 130) 1 | 26% (n = 124) | 13% (n = 245) | 33% (n = 138) |
C6 | 3% (n = 132) | 20% (n = 102) | 9% (n = 127) | 52% (n = 131) |
Target Locus 1 | sgRNA Recognition Sequence |
---|---|
C6 (chr6: 157,310,367–157,314,361) | sg1: GTGAGTGCACAC (22) 2 sg2: TGGGACACTATGATG (39) |
6T1_L (chr6: 168,378,356–168,380,872) | sg1: ACTCGGGCTGTG (35) sg2: CTGTGTGGGACT (26) |
6T1_R (chr6: 168,849,859–168,850,601) | sg1: GCAGAGGTGGCA (22) sg2: TGTGGGCAGAGG (20) |
6T2_L (chr6: 169,781,629–169,782,955 for sg1, and chr6: 169,803,533–169,807,849 for sg2) | sg1: ACCACTCGGAAA (21) sg2: GCTCTGTGTCTG (24) |
6T2_R (chr6: 170,500,882–170,504,181 for sg1, and chr6: 170,507,778–170,509,799 for sg2) | sg1: CTGCAGCCATCA (31) sg2: CACTCATTCAGC (26) |
4T (chr4: 186,033,845–186,035,464) | sg1: CCCTGAGGGATT (22) sg2: TCTGTACCCTGA (29) |
5T (chr5: 1,781,394–1,781,810) | sg1: AGGCTGAGGGTG (21) sg2: AGGGTGAGGCTG (23) |
22T (chr22: 47,211,081–47,213,579) | sg1: CATATTTGAGTG (56) sg2: GGACGGTCAGTG (30) |
Target | PCP-sfGFP Version | MCP-sfGFP Version | Proportion of Cells with Two Signals 2 | Signal-to-Background Ratio (Median) |
---|---|---|---|---|
C6 | 20% (n = 102) 1 | 52% (n = 131) | 34% | 2.3 |
6T1_L | 0% (n = 118) | 0% (n = 113) | 0% | n.a. |
6T1_R | 0% (n = 121) | 0% (n = 166) | 0% | n.a. |
6T2_L | 0% (n = 108) | 0% (n = 122) | 0% | n.a. |
6T2_R | 0% (n = 134) | 29% (n = 159) | 8% | 1.5 |
4T | 0% (n = 127) | 16% (n = 117) | 4% | 1.4 |
5T | 0% (n = 123) | 5% (n = 129) | 0% | 1.5 |
22T | 0% (n = 158) | 3% (n = 116) | 0% | 1.4 |
Repeat Cluster | Visualization Efficiency (MCP-sfGFP Version) | Number of sgRNA Repeats | Chromatin Status (ChromHMM18) | Transcription | Hi-C Compartment |
---|---|---|---|---|---|
IDR3 | 33% 1 | 45 | Quiescent | No | A |
C6 | 52% | 61 | Quiescent | Weak | A |
6T1_L | 0% | 61 | Quiescent | No | A |
6T1_R | 0% | 42 | Quiescent | No | A |
6T2_L_sg1 | 0% | 21 | Quiescent | No | A |
6T2_L_sg2 | 0% | 24 | Repressed Polycomb/weakly repressed Polycomb | No | A |
6T2_R_sg1 | 31% | 31 | Weakly Polycomb repressed | No | A |
6T2_R_sg2 | 0% | 26 | Quiescent | No | A |
4T | 16% | 51 | Quiescent | No | A |
5T | 5% | 44 | Quiescent | No | A |
22T | 3% | 86 | Quiescent | No | B |
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Viushkov, V.S.; Lomov, N.A.; Rubtsov, M.A. A Comparison of Two Versions of the CRISPR-Sirius System for the Live-Cell Visualization of the Borders of Topologically Associating Domains. Cells 2024, 13, 1440. https://doi.org/10.3390/cells13171440
Viushkov VS, Lomov NA, Rubtsov MA. A Comparison of Two Versions of the CRISPR-Sirius System for the Live-Cell Visualization of the Borders of Topologically Associating Domains. Cells. 2024; 13(17):1440. https://doi.org/10.3390/cells13171440
Chicago/Turabian StyleViushkov, Vladimir S., Nikolai A. Lomov, and Mikhail A. Rubtsov. 2024. "A Comparison of Two Versions of the CRISPR-Sirius System for the Live-Cell Visualization of the Borders of Topologically Associating Domains" Cells 13, no. 17: 1440. https://doi.org/10.3390/cells13171440