https://www.alphaknockout.com Mouse Robo3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Robo3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Robo3 gene (NCBI Reference Sequence: NM_001164767 ; Ensembl: ENSMUSG00000032128 ) is located on Mouse chromosome 9. 28 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 28 (Transcript: ENSMUST00000115038). Exon 8~11 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Robo3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-439O11 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous mutants display perinatal lethality, abnormal commissural axon growth, and fragile floor plates. Exon 8 starts from about 27.56% of the coding region. The knockout of Exon 8~11 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 1647 bp, and the size of intron 11 for 3'-loxP site insertion: 541 bp. The size of effective cKO region: ~1989 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 6 7 8 9 10 11 12 13 14 15 28 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Robo3 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(8489bp) | A(25.63% 2176) | C(22.83% 1938) | T(25.32% 2149) | G(26.22% 2226) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr9 - 37425629 37428628 3000 browser details YourSeq 217 2521 2942 3000 90.3% chr16 + 33387833 33388229 397 browser details YourSeq 201 2533 2942 3000 93.9% chr9 + 75450599 75451101 503 browser details YourSeq 199 2534 2837 3000 94.1% chr14 - 47591110 47591412 303 browser details YourSeq 192 2586 2942 3000 89.5% chr11 - 96845984 96846431 448 browser details YourSeq 186 2532 2929 3000 89.5% chr9 + 95538228 95538590 363 browser details YourSeq 186 2512 2723 3000 94.8% chr1 + 156676911 156677124 214 browser details YourSeq 184 2540 2941 3000 89.6% chr12 - 110378871 110379258 388 browser details YourSeq 181 2534 2788 3000 96.5% chr5 + 120216001 120216255 255 browser details YourSeq 181 2519 2723 3000 96.0% chr4 + 141137866 141138196 331 browser details YourSeq 180 2525 2942 3000 87.9% chr9 - 106486607 106486918 312 browser details YourSeq 178 2520 2723 3000 95.9% chr8 - 68058162 68058366 205 browser details YourSeq 178 2512 2723 3000 91.1% chr15 - 79274938 79275144 207 browser details YourSeq 178 2520 2725 3000 92.4% chr15 - 75952218 75952417 200 browser details YourSeq 178 2533 2724 3000 96.9% chr10 - 5113726 5113919 194 browser details YourSeq 178 2533 2722 3000 96.9% chr2 + 32867030 32867219 190 browser details YourSeq 178 2536 2871 3000 89.5% chr11 + 58089511 58089835 325 browser details YourSeq 176 2525 2722 3000 92.9% chr5 - 110631073 110631267 195 browser details YourSeq 176 2534 2737 3000 96.4% chr11 + 96092434 96092652 219 browser details YourSeq 176 2520 2742 3000 89.7% chr11 + 4710373 4710571 199 Note: The 3000 bp section upstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr9 - 37420640 37423639 3000 browser details YourSeq 65 2317 2421 3000 90.2% chr11 + 118291671 118291778 108 browser details YourSeq 64 2299 2421 3000 93.3% chr17 + 70018582 70018708 127 browser details YourSeq 57 2346 2411 3000 94.0% chr1 - 75536721 75536788 68 browser details YourSeq 57 2318 2415 3000 85.8% chr4 + 106874471 106874566 96 browser details YourSeq 56 2299 2392 3000 83.4% chr12 - 25279575 25279670 96 browser details YourSeq 56 2312 2412 3000 79.8% chr10 - 17611706 17611810 105 browser details YourSeq 55 2317 2419 3000 92.4% chr8 + 70052094 70052202 109 browser details YourSeq 55 2340 2421 3000 87.7% chr14 + 31881431 31881515 85 browser details YourSeq 53 2299 2377 3000 88.5% chr10 - 88889539 88889631 93 browser details YourSeq 53 2316 2415 3000 85.6% chr8 + 13701868 13701972 105 browser details YourSeq 53 2307 2410 3000 88.6% chr18 + 60685467 60685946 480 browser details YourSeq 53 2299 2377 3000 88.5% chr16 + 38848683 38848763 81 browser details YourSeq 52 2299 2415 3000 89.4% chr2 - 67610859 67610978 120 browser details YourSeq 52 2299 2377 3000 89.4% chr12 + 102882727 102882807 81 browser details YourSeq 51 2317 2409 3000 87.0% chr17 - 26711944 26712042 99 browser details YourSeq 49 2314 2385 3000 93.2% chr16 - 64897510 64897588 79 browser details YourSeq 49 2299 2365 3000 93.0% chr15 - 65841521 65841588 68 browser details YourSeq 49 2299 2415 3000 93.2% chr18 + 66592608 66592726 119 browser details YourSeq 48 2311 2377 3000 91.4% chr1 - 94610845 94610913 69 Note: The 3000 bp section downstream of Exon 11 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Robo3 roundabout guidance receptor 3 [ Mus musculus (house mouse) ] Gene ID: 19649, updated on 15-Oct-2019 Gene summary Official Symbol Robo3 provided by MGI Official Full Name roundabout guidance receptor 3 provided by MGI Primary source MGI:MGI:1343102 See related Ensembl:ENSMUSG00000032128 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Rig1; Rbig1; Rig-1; Robo3a; Robo3b Expression Biased expression in CNS E11.5 (RPKM 18.7), cortex adult (RPKM 8.7) and 5 other tissues See more Orthologs human all Genomic context Location: 9; 9 A4 See Robo3 in Genome Data Viewer Exon count: 30 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (37416045..37433436, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (37223630..37240760, complement) Chromosome 9 - NC_000075.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Robo3 ENSMUSG00000032128 Description roundabout guidance receptor 3 [Source:MGI Symbol;Acc:MGI:1343102] Gene Synonyms Rbig1, Rig-1, Rig1, Robo3a, Robo3b Location Chromosome 9: 37,415,669-37,433,246 reverse strand. GRCm38:CM001002.2 About this gene This gene has 6 transcripts (splice variants), 297 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Robo3- ENSMUST00000115038.2 4797 1402aa ENSMUSP00000110690.1 Protein coding CCDS52770 D3Z4M6 TSL:5 202 GENCODE basic APPRIS P2 Robo3- ENSMUST00000034643.11 4712 1380aa ENSMUSP00000034643.5 Protein coding - E9QPQ1 TSL:5 201 GENCODE basic APPRIS ALT2 Robo3- ENSMUST00000170512.7 5000 1345aa ENSMUSP00000150639.1 Nonsense mediated - A0A1L1SU85 TSL:1 205 decay Robo3- ENSMUST00000171467.7 3222 No - Retained intron - - TSL:1 206 protein Robo3- ENSMUST00000167089.7 2226 No - Retained intron - - TSL:2 203 protein Robo3- ENSMUST00000167216.1 668 No - Retained intron - - TSL:5 204 protein Page 6 of 8 https://www.alphaknockout.com 37.58 kb Forward strand 37.41Mb 37.42Mb 37.43Mb 37.44Mb Genes Robo4-205 >protein coding (Comprehensive set... Robo4-204 >nonsense mediated decay Robo4-203 >protein coding Robo4-202 >protein coding Robo4-201 >protein coding Robo4-206 >retained intron Contigs AC138284.9 > AC105958.11 > Genes (Comprehensive set... < Robo3-203retained intron < Robo3-202protein coding < Robo3-201protein coding < Robo3-206retained intron < Robo3-205nonsense mediated decay < Robo3-204retained intron Regulatory Build 37.41Mb 37.42Mb 37.43Mb 37.44Mb Reverse strand 37.58 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000115038 < Robo3-202protein coding Reverse strand 17.20 kb ENSMUSP00000110... MobiDB lite Low complexity (Seg) Superfamily Immunoglobulin-like domain superfamily Fibronectin type III superfamily SMART Immunoglobulin subtype Fibronectin type III Immunoglobulin subtype 2 Pfam PF13927 Fibronectin type III Immunoglobulin I-set PROSITE profiles Immunoglobulin-like domain Fibronectin type III PANTHER Roundabout homologue 3 PTHR12231 Gene3D Immunoglobulin-like fold CDD cd05726 cd00096 Fibronectin type III All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1402 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.