An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

Abstract

30We generated new genetic tools to efficiently tag genes in 31 Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions 32 to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 33 gene trapping in vivo based on CRE recombinase to avoid embryo 34 injections. DH significantly increases integration efficiency compared to 35 previous strategies and faithfully reports the expression pattern of genes 36 and proteins. The second technique targets genes lacking coding introns 37 using a two-step cassette exchange. First, we replace the endogenous 38 gene with an excisable compact dominant marker using CRISPR making a 39 null allele. Second, the insertion is replaced with a protein::tag cassette.40 2 This sequential manipulation allows the generation of numerous tagged 41 alleles or insertion of other DNA fragments that facilitates multiple 42 downstream applications. Both techniques allow precise gene manipulation 43 and facilitate detection of gene expression, protein localization and 44 assessment of protein function, as well as numerous other applications. 45 46 47 113 circular plasmid or can be circularized in vivo from an initial insertion locus 114 in the genome through Cre/loxP or Flp/FRT mediated recombination (Diao 115 et al., 2015; Nagarkar-Jaiswal et al., 2015b). Importantly, RMCE cassettes 116 can replace a SIC in either orientation with equal probability due to inverted 117 symmetric attP sequences. Therefore 50% of the insertions are inserted in 118 the opposite orientation of transcription and will not be included in the 119 4 mature mRNA. Hence, only half of all successful exchange events will 120 result in protein or gene trap lines. 121 Here, we show that by combining GFP protein traps and T2A-GAL4 122 gene traps in a single RMCE construct, named Double Header (DH), we 123 significantly increased the number of productive RMCE events for 124 MiMIC/CRIMIC containing genes to generate protein or gene trap alleles. 125 Importantly, we expand the ability to target SICs into genes regardless of 126 the presence of introns to allow access to virtually any gene in the fly 127 genome based on CRISPR/Cas9 mediated HDR. This provides a means to 128 create robust null alleles with simple screening, and to convert the SIC 129 insertion using any DNA, creating scarless modifications to facilitate 130 numerous downstream applications. 131 132 Results 133 134 Double Header (DH) improves the tagging rate of MiMIC containing 135 genes 136 137 SICs in coding introns can be converted into GFP protein traps or 138 T2A-GAL4 gene traps through RMCE. However, because RMCE of SICs in 139 MiMICs and CRIMICs can occur in either orientation, only one out of two 140 events produces a tag that is incorporated in the gene product. Moreover, 141 796 We thank the Bloomington Drosophila Stock Center and the Developmental 797 Studies Hybridoma Bank for antibodies. We thank Zelun Wang for technical 798 help, and Megan Campbell and Shinya Yamamoto for reading the 799 manuscript and providing helpful su...

Maternal almondex, a neurogenic gene, is required for proper subcellular Notch distribution in early Drosophila embryogenesis

“…Canton‐S was used as the wild‐type control strain. The genotypes of other Drosophila lines used were as follows: amx ΔCDS,ywing2+ , here called amx N to indicate that it is a null allele (Li‐Kroeger et al, ); Df(1)BSC663 (X:9,217,347…9,284,575), in which amx and nearby genes are uncovered by a molecularly defined deletion generated by FLP/FRT‐mediated recombination (Parks et al, ); pecanex 3 ( pcx 3 ), which has a loss‐of‐function pcx allele (Mohler & Carroll, ; Mohler, ); Protein disulfide isomerase ( Pdi )‐ GFP (a protein‐trap Pdi line) (Kelso et al, ); and a line carrying an approximately 3.3‐kbp genomic rescue construct containing wild‐type amx inserted onto a second chromosome phiC31 docking site (VK37), referred to as amx[+] (Jakobsdottir et al, ).…”

“…The amx N , a null mutant of amx , used in this study was reported before (Li‐Kroeger et al, ). In brief, we designed two guide RNAs (gRNAs) that targets 5ʹ and 3ʹ of the amx coding sequence (CDS) and deleted the amx CDS by replacing it with a yellow wing2+ marker using CRISPR‐mediated homology‐directed repair.…”

“…In this study, we took advantage of a clean null allele of amx , newly generated using CRISPR‐mediated homology‐directed repair, to understand the role of amx during early Drosophila embryogenesis (Li‐Kroeger et al, ). Our results confirm that amx is critical for lateral inhibition during neuroectoderm development, but we also found that amx is partially required for inductive signaling during mesectoderm development.…”

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

“…To generate attP-SA-sfGFP-SD-attP, a scaffold vector that we named pScaffold was produced by integrating annealed oligonucleotides with sequences M13For-attPfor-SbfI-AvrII-attPrev-M13rev in pCasper3 backbone in EcoRI-NotI sites. SA-sfGFP-SD was cloned as a threefragment ligation in pScaffold with linker-SA (amplified from pDoubleHeader (Li-Kroeger et al, 2018) with primers SA-for-Sbf and Linker-SA-rev_BamHI), sfGFP (amplified from pUAST-NLS-sfGFP-3XMyc-PEST with primers sfGFP-for_BamHI and sfGFP-rev_KpnI), and Linker-SD (amplified from pDoubleHeader with primers Linker-SD-for_KpnI and Linker-SD-rev_NotI).…”

“…SICs can also be used to generate conditional alleles of targeted genes (Flip-flop and FLPstop (Fisher et al, 2017;Nagarkar-Jaiswal et al, 2017). Finally, strategies have been developed to convert SICs through genetic crosses rather than by injection (Trojan Exons and Double Header;Nagarkar-Jaiswal et al, 2015b;Diao et al, 2015;Li-Kroeger et al, 2018).…”