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Mouse Rbp7 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rbp7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rbp7 gene (NCBI Reference Sequence: NM_022020 ; Ensembl: ENSMUSG00000028996 ) is located on Mouse chromosome 4. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000030848). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rbp7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-173D8 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 null mice have reduced retinyl ester levels in milk and other reductions in retinol and retinyl ester levels in a tissue and time dependent manner.

Exon 2 starts from about 18.41% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1343 bp, and the size of intron 3 for 3'-loxP site insertion: 2925 bp. The size of effective cKO region: ~1186 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 2 3 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Rbp7 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7686bp) | A(25.87% 1988) | C(22.34% 1717) | T(27.97% 2150) | G(23.82% 1831)

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% chr4 - 149453785 149456784 3000 browser details YourSeq 349 745 1701 3000 88.8% chr6 - 30012060 30012866 807 browser details YourSeq 304 743 1220 3000 93.0% chr1 + 74320021 75025687 705667 browser details YourSeq 281 730 1221 3000 93.3% chr11 + 79721181 80358884 637704 browser details YourSeq 272 777 1200 3000 93.1% chr10 - 62801764 63225361 423598 browser details YourSeq 271 1014 1353 3000 91.2% chr1 + 156695108 156695449 342 browser details YourSeq 266 1036 1358 3000 91.9% chr11 - 88967354 89010506 43153 browser details YourSeq 264 1036 1358 3000 91.6% chr6 - 128419000 128419336 337 browser details YourSeq 262 1036 1375 3000 88.4% chr1 - 169989253 169989584 332 browser details YourSeq 261 1036 1358 3000 92.9% chr10 - 100191229 100523975 332747 browser details YourSeq 257 753 1353 3000 89.8% chr4 - 152122280 152122858 579 browser details YourSeq 256 1027 1358 3000 92.5% chr10 - 82141761 82142114 354 browser details YourSeq 256 806 1217 3000 92.1% chr2 + 69637756 69638433 678 browser details YourSeq 255 730 1333 3000 86.9% chr17 + 15797574 15798135 562 browser details YourSeq 253 738 1351 3000 85.5% chr11 - 88921929 88922292 364 browser details YourSeq 250 1037 1351 3000 91.1% chr10 - 39206459 39206875 417 browser details YourSeq 248 1037 1358 3000 93.7% chr16 - 13956828 13957154 327 browser details YourSeq 246 1037 1353 3000 91.4% chr8 - 123120189 123120736 548 browser details YourSeq 245 1036 1358 3000 89.4% chr9 - 59646438 59646855 418 browser details YourSeq 244 777 1344 3000 94.5% chr2 - 119834614 119835180 567

Note: The 3000 bp section upstream of Exon 2 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% chr4 - 149449599 149452598 3000 browser details YourSeq 561 90 2258 3000 92.5% chr5 - 20866954 21166441 299488 browser details YourSeq 557 79 2254 3000 93.5% chr11 - 107128999 107183210 54212 browser details YourSeq 520 89 2260 3000 91.4% chr19 - 43219845 43685491 465647 browser details YourSeq 263 89 1197 3000 90.8% chr2 + 38974024 39093852 119829 browser details YourSeq 222 2062 2444 3000 90.3% chr1 + 191642281 191642684 404 browser details YourSeq 215 2066 2431 3000 91.9% chr5 - 147612911 147613578 668 browser details YourSeq 199 1048 1588 3000 88.7% chr16 + 30953308 30953960 653 browser details YourSeq 185 2010 2264 3000 89.6% chr19 + 57577878 57578100 223 browser details YourSeq 183 2100 2431 3000 87.5% chr10 - 62861249 62861548 300 browser details YourSeq 180 1085 1583 3000 83.5% chr1 - 131488727 131489142 416 browser details YourSeq 177 2058 2254 3000 95.9% chr11 - 115325998 115326215 218 browser details YourSeq 176 2062 2431 3000 85.8% chr8 + 25782314 25782735 422 browser details YourSeq 174 2047 2257 3000 90.3% chr8 - 36585124 36585318 195 browser details YourSeq 173 2034 2259 3000 90.7% chr16 + 93905613 93905826 214 browser details YourSeq 172 2001 2259 3000 89.0% chr6 + 86534845 86535081 237 browser details YourSeq 171 1328 1583 3000 91.0% chr7 - 126517479 126518050 572 browser details YourSeq 167 2062 2262 3000 90.0% chr7 + 127359478 127359667 190 browser details YourSeq 167 2062 2257 3000 93.1% chr12 + 3696770 3696963 194 browser details YourSeq 166 1115 1587 3000 88.4% chr16 - 20358694 20359182 489

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rbp7 retinol binding protein 7, cellular [ Mus musculus (house mouse) ] Gene ID: 63954, updated on 27-Aug-2019

Gene summary

Official Symbol Rbp7 provided by MGI Official Full Name retinol binding protein 7, cellular provided by MGI Primary source MGI:MGI:1890409 See related Ensembl:ENSMUSG00000028996 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as CRBP-III; 1110002J23Rik Expression Biased expression in subcutaneous fat pad adult (RPKM 3.4), heart adult (RPKM 2.0) and 13 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 E2 See Rbp7 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (149449687..149466462, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (148823810..148829077, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 3 transcripts

Gene: Rbp7 ENSMUSG00000028996

Description retinol binding protein 7, cellular [Source:MGI Symbol;Acc:MGI:1890409] Gene Synonyms 1110002J23Rik Location Chromosome 4: 149,449,687-149,454,978 reverse strand. GRCm38:CM000997.2 About this gene This gene has 3 transcripts (splice variants), 163 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rbp7- ENSMUST00000030848.2 619 134aa ENSMUSP00000030848.2 Protein coding CCDS18959 Q540P4 TSL:1 201 Q9EPC5 GENCODE basic APPRIS P1

Rbp7- ENSMUST00000148596.7 507 68aa ENSMUSP00000119166.1 Nonsense mediated - F6R1Y3 CDS 5' 203 decay incomplete TSL:3

Rbp7- ENSMUST00000132103.1 416 No - lncRNA - - TSL:3 202 protein

25.29 kb Forward strand 149.44Mb 149.45Mb 149.46Mb Genes Trmt112-ps2-201 >processed pseudogene (Comprehensive set...

Contigs AL606973.29 > Genes (Comprehensive set... < Rbp7-201protein coding

< Rbp7-203nonsense mediated decay

< Rbp7-202lncRNA

Regulatory Build

149.44Mb 149.45Mb 149.46Mb Reverse strand 25.29 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript pseudogene

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Transcript: ENSMUST00000030848

< Rbp7-201protein coding

Reverse strand 5.29 kb

ENSMUSP00000030... Superfamily Calycin Prints Cytosolic fatty-acid binding Pfam Lipocalin/cytosolic fatty-acid binding domain PROSITE patterns Cytosolic fatty-acid binding PANTHER PTHR11955:SF97

Intracellular lipid binding protein Gene3D Calycin

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 134

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

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