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| Status |
Public on Oct 09, 2023 |
| Title |
Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [Screen] |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
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| Summary |
CRISPR-Cas13 systems have been adapted as versatile toolkits for RNA-related applications. Here we systematically evaluate the performance of several prominent Cas13 family effectors (Cas13a, Cas13b and Cas13d) under lentiviral vectors and reveal surprisingly differential defects and characteristics of these systems. Using RNA immunoprecipitation sequencing, transcriptome profiling, biochemistry analysis and high-throughput CRISPR-Cas13 screening approaches, we determine that each Cas13 system has its intrinsic RNA targets in mammalian cells. Viral process-related host genes can be targeted by Cas13 and affect the production of fertile lentiviral particles, thereby restricting the utility of lentiviral Cas13 systems. Multiple RNase activities of Cas13 are involved in endogenous RNA targeting. Unlike target-induced collateral effect, intrinsic RNA targeting can be specific, target-independent and dynamically tuned by varied states of Cas13 nucleases. Our work not only provides guidance to appropriately utilize lentiviral Cas13 systems, but also raises cautions about intrinsic RNA targeting during Cas13-based basic and therapeutic applications.
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| Overall design |
Cas9 or Cas13 expressing vector transiently transfected 293FT cells were used for RIP-seq library construction or RNA-seq library construction and deep sequencing. The crRNA lentiviral library-infected cas13d/b-expressing A375 cells harvested in day 5 and day 33 were used for DNA extraction, gRNA enrichment by PCR amplification and deep sequencing.
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| Contributor(s) |
Li Z, Li Z, Cheng X, Li W, Fei T |
| Citation(s) |
37872368 |
| |
| Submission date |
May 12, 2022 |
| Last update date |
Oct 25, 2023 |
| Contact name |
Teng Fei |
| E-mail(s) |
feiteng@mail.neu.edu.cn
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| Phone |
86-24-83656103
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| Organization name |
Northeastern University, China
|
| Department |
College of Life and Health Sciences
|
| Lab |
Teng Fei's lab
|
| Street address |
195 Chuangxin Rd. Life Sciences Building A426. Hunnan District.
|
| City |
Shenyang |
| State/province |
Liaoning |
| ZIP/Postal code |
110169 |
| Country |
China |
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| Platforms (3) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL26526 |
Illumina NovaSeq 6000 (synthetic construct) |
| GPL26697 |
HiSeq X Ten (synthetic construct) |
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| Samples (29)
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| This SubSeries is part of SuperSeries: |
| GSE202899 |
Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems |
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| Relations |
| BioProject |
PRJNA837526 |
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