Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3
Figure Lengend Snippet: CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif
Article Snippet: Construction of robo3TcRobo2/3 donor plasmid The initial robo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611).
Techniques: CRISPR, Plasmid Preparation, Modification, Polymerase Chain Reaction, Sequencing