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Precise RNA knockdown of smn1,or tardbp and tarbpl by CRISPR-QKD and transcription activation of lin28a and sox9b by CRISPR-Qa in zebrafish

ABSTRACT: CRISPR-Cas systems have revolutionized gene regulation technologies in various organisms, including zebrafish. However, most zebrafish studies rely on transient injections of CRISPR components, with limited use of transgenic models, primarily for Cas9-mediated knockouts. This is largely due to challenges in achieving sustained and effective expression of Cas effectors. To address these challenges, we introduce the CRISPR-Q system, which integrates the QFvpr/QUAS binary expression system with CRISPR-Cas effectors. This approach overcomes limitations associated with transient mRNA or protein delivery and circumvents the toxicity and silencing issues typical of other binary systems, such as Gal4/UAS. The CRISPR-Q system enables robust expression of CasRx or dCas9vpr, facilitating efficient transcript knockdown (CRISPR-QKD) and gene activation (CRISPR-Qa). Using this system, we successfully achieved significant knockdown of smn1 and the simultaneous knockdown of the paralogs tardbp and tardbpl, modeling phenotypes of spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), respectively. Furthermore, CRISPR-Qa effectively activated the endogenous genes lin28a and sox9b, demonstrating the system's broad applicability. The CRISPR-Q system represents a significant advancement in zebrafish genetic manipulation, providing a robust and versatile platform for studying gene function and modeling human diseases, with potential extensions to other model organisms

ORGANISM(S): Danio rerio

PROVIDER: GSE268371 | GEO | 2024/06/25

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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Precise RNA knockdown of smn1,or tardbp and tarbpl by CRISPR-QKD and transcription activation of lin28a and sox9b by CRISPR-Qa in zebrafish

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