Single-Cell Cas12a Platform for Screening and CRISPRi
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE283574 | GEO | 2026/04/07
REPOSITORIES: GEO
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Project description:Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes or an oncogenic translocation. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. Efficiency and TSG combos These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo.
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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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