Generation of a Syt7 floxed allele. a Diagrammatic representation of the genomic sequence with the Syt7 critical exon highlighted, the corresponding template for lssDNA synthesis and the position of sgRNAs for in vivo delivery together with the primer locations used for reverse transcription and for genotyping. Note loxP sites in the lssDNA prevent reprocessing of repaired alleles by CRISPR-Cas9 complex. Diagram shows the process for the generation of lssDNA through in vitro transcription and reverse transcription. HA homology arm. b PCR products amplified from genomic DNA extracted from the 17 F0 born from the microinjection session using Syt7-F1 and Syt7-R1 primers. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). Sequence trace data derived from animals Syt7-4 and Syt7-8 are displayed in Additional file 2: Figure S1.