1020B Poster - 16. Techniques and technology
Friday April 08, 2:00 PM - 4:00 PM

Authors:
Oguz Kanca ^1,2; Jonathan Zirin ³; Yanhui Hu ³; Burak Tepe ^1,2; Debdeep Dutta ^1,2; Wen-Wen Lin ^1,2; Liwen Ma ^1,2; Ming Ge ^1,2; Zhongyuan Zuo ^1,2; Lu-Ping Liu ³; Robert W. Levis ⁴; Norbert Perrimon ^3,6; Hugo J. Bellen ^1,5

Affiliations:
1) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX ; 2) Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX; 3) Department of Genetics Harvard Medical School, Boston, MA ; 4) Department of Embryology, Carnegie Institution for Science, Baltimore, MD ; 5) Department of Neuroscience, Baylor College of Medicine, Houston, TX ; 6) Howard Hughes Medical Institute, Harvard Medical School, Boston, MA

Keywords:
g. CRISPR/Cas9; e. gene targeting and modification

Gene traps and protein traps are valuable genetic tools to study gene function. The majority of these constructs are inserted in the genome as artificial exons, with a Splice Acceptor (SA)-effector-Splice Donor (SD) configuration. The artificial exon must be inserted in an intron between two coding exons to function as a gene or protein trap. About 50% of the Drosophila genes do not have a suitable coding intron. We developed methods to replace the coding region of these genes with a Kozak-GAL4-3XP3GFP cassette creating a null allele, while expressing GAL4. We have generated >100 KozakGAL4 alleles to date. Analysis of the expression patterns in 3^rd instar larval brains shows that ~80% of the tested KozakGAL4 alleles can drive expression of UAS reporter genes with discernible patterns. We selected KozakGAL4 alleles that drive reporter expression in restricted patterns and used publicly available single cell RNA sequencing (scRNAseq) data to find other genes that are co-expressed with the targeted gene. T2AGAL4 alleles of such genes, identified by scRNAseq, drive expression of reporter genes in patterns overlapping with the KozakGAL4 targeted genes. scRNAseq data serve as an independent means to verify the accuracy of KozakGAL4 expression patterns for reporting the expression pattern of the targeted gene. Hence, KozakGAL4 alleles serve as an alternative approach to artificial exon-based methods for genes lacking a suitable intron.
We also designed and generated new homology donor vector backbones to facilitate the generation of homology donor constructs. We showed that in vivo linearization of the donor constructs improves the efficiency of incorporation of large constructs (>5kb) using short homology arms (200 bps). Short homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps in donor construct generation. We have designed new vector backbones that include both gene specific sgRNA(s) and an sgRNA to linearize the homology donor together with the homology arms. This strategy obviates cloning separate sgRNAs and ensures delivery of all the components for targeting a gene in a single plasmid. We show that using this strategy greatly facilitates cloning of the donor constructs and increases the successful transgenesis rate to 80%. These upgrades, together with the KozakGAL4 strategy, will enable efficient targeting of 80% of the conserved fly genes by the Gene Disruption Project.