AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma

Abstract

A causative understanding of genetic factors that regulate glioblastoma (GBM) pathogenesis is of central importance. Here, we developed an adeno-associated virus (AAV)-mediated autochthonous CRISPR screen in GBM. Stereotaxic delivery of an AAV library targeting genes commonly mutated in human cancers into the brains of conditional Cas9 mice resulted in tumors that recapitulate human GBM. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlate with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as Mll2, B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express Homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo.

Acceleration of cancer science with genome editing and related technologies

“…[41][42][43] In addition, adeno-associated virus (AAV) vector-mediated direct delivery of pooled sgRNA library enabled bona fide in vivo screening, although the capacity of the library size was tightly restricted in this strategy ( Figure 4B, top arrow). 44,45 Such ex vivo and in vivo screening studies were nicely summarized recently by Chow and Chen. 46 The second study is a series of studies reporting long non-coding RNA (lncRNA) screening.…”

“…After this publication, various related studies depending on the transplantation‐based approach have been conducted . In addition, adeno‐associated virus (AAV) vector‐mediated direct delivery of pooled sgRNA library enabled bona fide in vivo screening, although the capacity of the library size was tightly restricted in this strategy (Figure B, top arrow) . Such ex vivo and in vivo screening studies were nicely summarized recently by Chow and Chen …”

Testing thousands of nanoparticles in vivo using DNA barcodes

“…Similar approaches have been used to identify genes that suppress primary tumor growth in the liver and brain (Fig. 4B) [53,54]. Scientists also designed a combinatorial DNA barcode system named ‘PolyLox’ that identified stem and progenitor cells that led to the development of the immune system [55].…”

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome

“…Pooled screens have also enabled high-throughput genetic studies in vivo , which can include viral infection of CRISPR reagents ex vivo with subsequent transplantation (Chen et al, 2015; Manguso et al, 2017) or direct delivery of CRISPR reagents in vivo (Chow et al, 2017). Delivery of both the gRNA and nuclease in a single vector is preferable for in vivo applications to achieve higher transduction rates as opposed to a split vector system (i.e., separate vector for nuclease and gRNA) requiring co-transduction (Ran et al, 2015; Xue et al, 2014).…”

“…Libraries are prepared for deep sequencing by PCR from genomic DNA using primers specific to the genomically integrated CRISPR construct or using locus-specific primers for non-integrating library delivery methods, such as AAV (Chow et al, 2017; Wang et al, 2017). Sequencing reads are aligned to the original gRNA library, resulting in read counts for each gRNA in the library before and after phenotypic selection.…”