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| Status |
Public on Dec 06, 2020 |
| Title |
Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for circRNA functional characterization. To address this issue, we leveraged the CRISPR/Cas13d system2 for functional study of circRNAs by optimizing the strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNA screening yielded a high rate of false positive phenotypes. Furthermore, using a CRISPR/Cas13d screening library targeting over 2,500 human hepatocellular carcinomas (HCC) related circRNAs, we identified a group of circRNAs, whose inhibition increased the therapeutic efficacy of sorafenib, an approved HCC drug. Collectively, these data demonstrate that CRISPR/Cas13d system is an effective approach to study the function of circRNAs in a high-throughput manner.
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| Overall design |
Identification of circular RNAs essential for cell survival in hepatocellular carcinoma using CRISPR RNA Cas13d and shRNA screening. CRISPR Cas13d screening was also performed to identify circular RNAs that drive resistance to sorafenib in liver cancer. Comparative analysis on specificity of CRISPR Cas13d and shRNA in knocking circular RNA was also performed using RNA-Seq
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| Contributor(s) |
Zhang Y, Nguyen TM, Zhang X, Wang L, Phan T, Clohessy JG, Pandolfi P |
| Citation(s) |
33478577 |
| |
| Submission date |
Dec 05, 2020 |
| Last update date |
Jan 27, 2021 |
| Contact name |
Pier Paolo Pandolfi |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Torino
|
| Street address |
8 Via Verdi
|
| City |
Torino |
| State/province |
TO |
| ZIP/Postal code |
10124 |
| Country |
USA |
| |
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| Platforms (4)
|
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
|
| Samples (22)
|
| GSM4957917 |
shRNA_essential_circRNA_screen_Day0_rep1 |
| GSM4957918 |
shRNA_essential_circRNA_screen_Day0_rep2 |
| GSM4957919 |
shRNA_essential_circRNA_screen_Day14_rep1 |
| GSM4957920 |
shRNA_essential_circRNA_screen_Day14_rep2 |
| GSM4957921 |
sgRNA_essential_circRNA_screen_Day0_rep1 |
| GSM4957922 |
sgRNA_essential_circRNA_screen_Day0_rep2 |
| GSM4957923 |
sgRNA_essential_circRNA_screen_Day14_rep1 |
| GSM4957924 |
sgRNA_essential_circRNA_screen_Day14_rep2 |
| GSM4957925 |
shRNA_essential_circRNA_screen_Day0_deep_sequencing_rep1 |
| GSM4957926 |
shRNA_essential_circRNA_screen_Day0_deep_sequencing_rep2 |
| GSM4957927 |
shRNA_essential_circRNA_screen_Day14_deep_sequencing_rep1 |
| GSM4957928 |
shRNA_essential_circRNA_screen_Day14_deep_sequencing_rep2 |
| GSM4957929 |
sgRNA_sorafenib_resistant_circRNA_screen_DMSO_rep1 |
| GSM4957930 |
sgRNA_sorafenib_resistant_circRNA_screen_DMSO_rep2 |
| GSM4957931 |
sgRNA_sorafenib_resistant_circRNA_screen_Sorafenib_rep1 |
| GSM4957932 |
sgRNA_sorafenib_resistant_circRNA_screen_Sorafenib_rep2 |
| GSM4957933 |
circGFP-targeting_gRNA_RNASeq_rep1 |
| GSM4957934 |
circGFP-targeting_gRNA_RNASeq_rep2 |
| GSM4957935 |
Non-targeting_gRNA_RNASeq |
| GSM4957936 |
circGFP-targeting_shRNA_RNASeq_rep1 |
| GSM4957937 |
circGFP-targeting_shRNA_RNASeq_rep2 |
| GSM4957938 |
Non-targeting_shRNA_RNASeq |
|
| Relations |
| BioProject |
PRJNA682745 |
| SRA |
SRP296397 |
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