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Next Generation Sequencing Facilitates Transcriptomes Quantitative Analysis of HEK293T cells transfected with the plasmids respectively encoding RfxCas13d, crRNA and NeuN.

ABSTRACT: To determine what effect the collateral activity of RfxCas13d has on HEK293T cells upon targeting overexpressed NeuN. The plasmids encoding RfxCas13d, NeuN and NT/targeting crRNA into HEK293T cells. After 24 hours of transfection, cells were collected and extracted for total RNA. Then, we performed RNA-seq to compare the differentially expressed genes between cells transfected with targeting crRNAs and non-targeting crRNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE222451 | GEO | 2023/01/14

REPOSITORIES: GEO

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Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism. 4 Samples: Protein-associated small RNAs

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Next Generation Sequencing Facilitates Transcriptomes Quantitative Analysis of HEK293T cells transfected with the plasmids respectively encoding RfxCas13d, crRNA and NeuN.

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