Minimized combinatorial CRISPR screens identify genetic interactions in autophagy
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ABSTRACT: Development of the 3Cs multiplex technology for combinatorial screening and identification of genetic interactions among human autophagy genes in cell proliferation and autophagy flux.
Project description:CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a system (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.
Project description:Autophagy, a critical process for the vacuolar degradation of proteins and organelles, is governed by multiple conserved autophagy-related (ATG) proteins. The central component of the ATG machinery is the ubiquitin-like protein ATG8, which is essential for multiple steps of the autophagy process, including phagophore expansion, autophagosome closure, trafficking and fusion with the lysosome/vacuole, and selective cargo recruitment. Currently, our understanding of the roles of ATG8 in plant autophagy and the functional specialization of ATG8 family members is limited due to genetic redundancy. To assess the roles of ATG8 genes in plant autophagy, here we used CRISPR/Cas9 technology to systematically knockout the Arabidopsis ATG8 genes. By analyzing the atg8 mutants, we found that in contrast to mammalian ATG8s, in which the LC3s and GABARAP subfamilies play distinct roles in the autophagic process, Arabidopsis ATG8s perform an overlapping function in controlling autophagic flux. Combinatorial mutations of Clade I and Clade II ATG8s resulted in severely impaired autophagy under nutrient-starved conditions. Furthermore, we found that RABG3 proteins, members of the RAB7/RABG GTPase family, interact with ATG8s through AIM-LDS interfaces, and that such interaction is essential for the association of RABG3 proteins with the autophagosomal membrane and probably for the fusion of autophagosome with the vacuole, but is not required for endosomal trafficking. With the collection of multiple high-order atg8 mutants generated in this study, we now provide a venue to study the roles of ATG8 genes in canonical autophagy and non-canonical autophagy in Arabidopsis.
Project description:Murine pancreatic cancer cells (HY15549 cells) established from genetically engineered mouse models (GEMM) of PDAC (p48-Cre+, KrasLSL-G12D/+, Trp53lox/+; KPC mice) were transfected with the GFP-LC3-RFP autophagy flux reporter from Mizushima lab, wherein reduction in the GFP/RFP ratio indicates increase in autophagy flux. Organoids derived from these cells were dissociated into single cells and sorted into autophagy-high (AThi) and autophagy-low (ATlo) populations according to GFP/RFP signal ratio. To validate this reporter, RNA was extracted from these two populations and expression of autophagy/lysosome related genes was examined.
Project description:Autophagy is a cellular recycling process that can promote tumor growth, anti-tumor immune response, and resistance to therapy in colorectal cancer (CRC). Here, we show that small GTPase Rab27B, a known regulator of vesicle trafficking and extracellular vesicle secretion, to control the autophagy process in CRC. Depletion of Rab27B in CRC cells showed an abnormal accumulation of autophagy vesicles and increased autophagy markers, indicating a defect in autophagy flux. Imaging analysis indicated that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. Loss of Rab27B significantly impacted CRC cell growth in both in vitro 3D growth and in vivo tumorigenesis studies. Together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC and identify Rab27B as a potential therapeutic target for CRC.