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Mouse Robo4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Robo4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Robo4 gene (NCBI Reference Sequence: NM_028783 ; Ensembl: ENSMUSG00000032125 ) is located on Mouse chromosome 9. 18 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000102895). Exon 8~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Robo4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-356D13 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: Mice homozygous for a reporter/null allele display enhanced VEGF-induced endothelial migration, tube formation and vascular permeability, and show increased pathologic angiogenesis and vascular leak in models of oxygen-induced retinopathy and choroidal neovascularization.

Exon 8 starts from about 38.85% of the coding region. The knockout of Exon 8~10 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 689 bp, and the size of intron 10 for 3'-loxP site insertion: 1706 bp. The size of effective cKO region: ~1159 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 7 8 9 10 11 12 18 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Robo4 Homology arm cKO region loxP site

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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(7659bp) | A(25.06% 1919) | C(25.02% 1916) | T(25.46% 1950) | G(24.47% 1874)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

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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 + 37402324 37405323 3000 browser details YourSeq 55 1451 1523 3000 92.1% chr4 + 137988512 137988935 424 browser details YourSeq 49 1318 1494 3000 78.0% chr11 - 19430353 19430524 172 browser details YourSeq 49 1457 1547 3000 93.2% chr4 + 86130228 86130347 120 browser details YourSeq 48 1433 1493 3000 91.4% chr4 + 125059878 125059943 66 browser details YourSeq 46 1424 1489 3000 84.9% chr12 + 25277562 25277627 66 browser details YourSeq 45 1424 1488 3000 84.7% chr1 - 86257739 86257803 65 browser details YourSeq 44 1 45 3000 100.0% chr3 - 7016763 7016887 125 browser details YourSeq 41 1432 1483 3000 91.2% chr14 - 65160583 65160633 51 browser details YourSeq 41 16 61 3000 88.7% chr15 + 102716413 102716456 44 browser details YourSeq 40 1445 1493 3000 91.9% chr12 - 54229158 54229216 59 browser details YourSeq 38 1424 1487 3000 79.7% chr11 - 100357238 100357301 64 browser details YourSeq 37 1445 1486 3000 95.3% chr4 - 45223580 45223622 43 browser details YourSeq 36 1436 1482 3000 92.2% chr11 - 40750063 40750108 46 browser details YourSeq 36 1442 1487 3000 89.2% chr2 + 145635954 145635999 46 browser details YourSeq 36 1436 1494 3000 92.9% chr11 + 114524551 114524848 298 browser details YourSeq 34 1448 1483 3000 97.3% chr2 - 28786044 28786079 36 browser details YourSeq 34 1436 1482 3000 89.5% chr2 + 168800943 168800988 46 browser details YourSeq 34 1400 1483 3000 94.8% chr17 + 45019759 45019844 86 browser details YourSeq 34 1436 1484 3000 94.8% chr1 + 172437336 172437386 51

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 + 37406483 37409482 3000 browser details YourSeq 31 2317 2360 3000 97.0% chr18 - 39520915 39521105 191 browser details YourSeq 30 375 420 3000 96.9% chr9 - 97173367 97173438 72 browser details YourSeq 30 2892 2936 3000 94.2% chr5 + 132416275 132416320 46 browser details YourSeq 22 376 421 3000 74.0% chr12 - 100846342 100846387 46 browser details YourSeq 21 570 590 3000 100.0% chr2 - 123490341 123490361 21 browser details YourSeq 21 1946 1966 3000 100.0% chr1 - 172791311 172791331 21

Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Robo4 roundabout guidance receptor 4 [ Mus musculus (house mouse) ] Gene ID: 74144, updated on 12-Aug-2019

Gene summary

Official Symbol Robo4 provided by MGI Official Full Name roundabout guidance receptor 4 provided by MGI Primary source MGI:MGI:1921394 See related Ensembl:ENSMUSG00000032125 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 AI593217; 1200012D01Rik Expression Broad expression in lung adult (RPKM 31.0), subcutaneous fat pad adult (RPKM 26.6) and 19 other tissuesS ee more Orthologs human all

Genomic context

Location: 9; 9 A4 See Robo4 in Genome Data Viewer

Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (37401902..37414023)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (37209631..37221607)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 6 transcripts

Gene: Robo4 ENSMUSG00000032125

Description roundabout guidance receptor 4 [Source:MGI Symbol;Acc:MGI:1921394] Gene Synonyms 1200012D01Rik, Magic roundabout Location Chromosome 9: 37,401,897-37,415,115 forward strand. GRCm38:CM001002.2 About this gene This gene has 6 transcripts (splice variants), 99 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

Robo4- ENSMUST00000214185.1 4952 1022aa ENSMUSP00000150722.1 Protein coding CCDS80976 A0A1L1SUF8 TSL:1 205 GENCODE basic APPRIS ALT2

Robo4- ENSMUST00000102895.5 3226 1015aa ENSMUSP00000099959.4 Protein coding CCDS22977 A0A0R4J197 TSL:1 201 GENCODE basic APPRIS P3

Robo4- ENSMUST00000115046.7 3878 1074aa ENSMUSP00000110698.1 Protein coding - D3Z4M4 TSL:2 202 GENCODE basic APPRIS ALT2

Robo4- ENSMUST00000115048.8 3392 911aa ENSMUSP00000110700.2 Protein coding - E9QN68 TSL:1 203 GENCODE basic

Robo4- ENSMUST00000156972.1 3947 318aa ENSMUSP00000150053.1 Nonsense mediated - A0A1L1SSV2 TSL:2 204 decay

Robo4- ENSMUST00000215777.1 530 No - Retained intron - - TSL:2 206 protein

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33.22 kb Forward strand 37.40Mb 37.41Mb 37.42Mb Genes (Comprehensive set... Robo4-205 >protein coding

Robo4-204 >nonsense mediated decay

Robo4-203 >protein coding

Robo4-202 >protein coding

Robo4-201 >protein coding

Robo4-206 >retained intron

Contigs AC138284.9 > Genes < Robo3-203retained intron (Comprehensive set...

< Robo3-202protein coding

< Robo3-201protein coding

< Robo3-206retained intron

< Robo3-205nonsense mediated decay

< Robo3-204retained intron

Regulatory Build

37.40Mb 37.41Mb 37.42Mb Reverse strand 33.22 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000102895

11.51 kb Forward strand

Robo4-201 >protein coding

ENSMUSP00000099... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily

Fibronectin type III superfamily SMART Immunoglobulin subtype Fibronectin type III

Immunoglobulin subtype 2 Pfam PF13927 Immunoglobulin I-set Fibronectin type III

PROSITE profiles Fibronectin type III

Immunoglobulin-like domain PANTHER PTHR44170

PTHR44170:SF11 Gene3D Immunoglobulin-like fold CDD Fibronectin type III

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend frameshift variant missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1015

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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