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New High-Throughput AAV CRISPR Screening Method Could Enable Study of Diverse Tissue Types

NEW YORK — Using adeno-associated viruses (AAV), researchers have developed a new platform for in vivo CRISPR screening that could facilitate a better understanding of disease-causing genes and their phenotypes in various cell types. 

In a study published in Nature on Wednesday, a team led by scientists at ETH Zurich described AAV-Perturb-seq, which they specifically applied to understand the phenotype of genes linked to a genetic disorder called DiGeorge or 22q11.2 deletion syndrome, in a mouse model. Deletions in chromosome 22 cause this condition and affect one in 4,000 births, often leading to heart problems, immune deficiency, and cognitive problems, among other symptoms.

Current CRISPR screening methods, which mainly use lentivirus, are restricted to in vitro applications and only work for tissues that this virus can infect, the authors noted. They pointed out an urgent need for direct in vivo single-cell screens that can be used on different tissue types, a reason why they chose AAVs, which can be delivered intravenously, targeting a wide range of tissue and cell types.

"AAV-Perturb-seq offers the opportunity to directly examine multiple genes in several cell types at the single-cell level in the same animal without restriction to tissue or developmental time points, opening further possibilities for studying health and disease processes in vivo," the authors wrote.

The researchers began by creating an AAV guide RNA (gRNA) library focused on genes located within the human 22q11.2 locus that are conserved in the mouse genome. They identified 29 genes expressed in the adult mouse cortex for which they designed the gRNA sequences.

Next, they injected the recombinant AAVs containing the gRNA into LSL-Cas9 mice or conducted transcriptional inhibition in dCas9-KRAB mice and collected brain samples three weeks later. The isolated nuclei from various cells were subjected to clustering analysis, which showed the AAVs had infected neuronal and non-neuronal brain cells.

Their findings showed the perturbation of genes DGCR8, DGCR14, GNB1L, and UFD1L explained approximately 40 percent of the transcriptional changes observed in a 22q11.2-deletion mouse model. However, none of the genes could explain the predisposition of people with the condition for neurodevelopmental and psychiatric disorders.

While the function of each gene in mature brain cells is poorly understood and has never been systematically investigated, the researchers identified connections between DGCR14 and GNB1L related to adult neuron physiology relevant to 22q11.2DS pathology.

"Overall, our results indicate that DGCR8, DGCR14, and GNB1L may contribute to 22q11.2DS by broadly altering the expression of genes associated with disease susceptibility in vivo, emerging after development and through a mechanism that involves RNA regulation in mature neurons," the authors concluded.

The next steps, they said, would be to determine whether 22q11.2DS-associated neuronal and cognitive phenotypes are salvaged by restoring DGCR8, DGCR14, GNB1L, and UFD1L expression during or after development.