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Mouse Crabp1 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Crabp1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Crabp1 gene (NCBI Reference Sequence: NM_013496 ; Ensembl: ENSMUSG00000032291 ) is located on Mouse chromosome 9. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000034830). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Crabp1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-393C21 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: Homozygotes for targeted null mutations are phenotypically normal and fertile.
Exon 2 starts from about 17.27% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 511 bp, and the size of intron 2 for 3'-loxP site insertion: 2722 bp. The size of effective cKO region: ~679 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 2 4 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Homology arm Exon of mouse Crabp1 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(7179bp) | A(24.91% 1788) | C(24.72% 1775) | T(24.4% 1752) | G(25.96% 1864)
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.
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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 + 54762234 54765233 3000 browser details YourSeq 41 846 975 3000 95.8% chr2 - 178288694 178288823 130 browser details YourSeq 38 281 354 3000 97.6% chr1 - 59582851 59583189 339 browser details YourSeq 37 263 306 3000 93.2% chr11 + 5176537 5176581 45 browser details YourSeq 35 263 305 3000 90.7% chrX - 102610939 102610981 43 browser details YourSeq 33 266 356 3000 68.2% chr1 + 119860791 119860881 91 browser details YourSeq 31 2620 2680 3000 97.1% chr12 - 19383808 19383868 61 browser details YourSeq 31 2620 2680 3000 97.1% chr12 + 23647300 23647360 61 browser details YourSeq 31 2620 2680 3000 97.1% chr12 + 23808339 23808399 61 browser details YourSeq 31 2620 2680 3000 97.1% chr12 + 23193415 23193475 61 browser details YourSeq 31 2620 2680 3000 97.1% chr12 + 23014165 23014225 61 browser details YourSeq 31 2620 2680 3000 97.1% chr12 + 22748901 22748961 61 browser details YourSeq 31 2620 2680 3000 97.1% chr12 + 22479609 22479669 61 browser details YourSeq 29 84 121 3000 90.4% chr5 - 115806088 115806124 37 browser details YourSeq 29 265 306 3000 90.4% chr2 - 129388851 129388891 41 browser details YourSeq 27 2620 2675 3000 86.3% chr1 - 141943354 141943407 54 browser details YourSeq 27 2620 2675 3000 86.3% chr18 + 29300203 29300256 54 browser details YourSeq 24 847 882 3000 83.4% chr6 - 42683339 42683374 36 browser details YourSeq 24 763 794 3000 80.7% chr17 + 32098751 32098781 31 browser details YourSeq 23 2107 2145 3000 79.5% chr14 + 93912746 93912784 39
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% chr9 + 54765913 54768912 3000 browser details YourSeq 123 381 530 3000 95.0% chr8 + 57442828 57443080 253 browser details YourSeq 122 381 539 3000 90.3% chr10 - 93149912 93150062 151 browser details YourSeq 119 383 539 3000 94.3% chr8 - 47121993 47122253 261 browser details YourSeq 106 382 533 3000 93.5% chr12 + 62661243 62661400 158 browser details YourSeq 104 398 538 3000 93.1% chr10 + 62676764 62677062 299 browser details YourSeq 98 383 539 3000 94.7% chrX - 13222831 13222990 160 browser details YourSeq 93 383 527 3000 91.3% chr8 - 126290615 126290787 173 browser details YourSeq 90 383 876 3000 76.2% chr10 - 90626703 90626858 156 browser details YourSeq 86 367 539 3000 81.8% chr11 + 47410170 47410264 95 browser details YourSeq 85 382 520 3000 80.2% chr5 - 124783038 124783149 112 browser details YourSeq 82 440 545 3000 95.6% chr11 - 53112221 53112519 299 browser details YourSeq 82 409 536 3000 89.1% chr10 - 63694765 63694881 117 browser details YourSeq 79 431 537 3000 96.6% chr11 - 17015868 17015990 123 browser details YourSeq 78 409 518 3000 93.6% chr11 + 24289780 24289942 163 browser details YourSeq 75 381 534 3000 76.7% chr11 - 52988692 52988785 94 browser details YourSeq 74 395 906 3000 74.8% chr2 - 116899242 116899544 303 browser details YourSeq 73 465 543 3000 97.5% chr3 - 119413107 119413265 159 browser details YourSeq 73 383 521 3000 81.2% chr1 - 92073122 92073227 106 browser details YourSeq 71 459 539 3000 94.7% chr11 + 25210541 25210620 80
Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Crabp1 cellular retinoic acid binding protein I [ Mus musculus (house mouse) ] Gene ID: 12903, updated on 14-Aug-2019
Gene summary
Official Symbol Crabp1 provided by MGI Official Full Name cellular retinoic acid binding protein I provided by MGI Primary source MGI:MGI:88490 See related Ensembl:ENSMUSG00000032291 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 Rbp-5; CrabpI; CRABP-I; Crabp-1; AI326249 Expression Biased expression in CNS E11.5 (RPKM 232.5), limb E14.5 (RPKM 196.0) and 3 other tissues See more Orthologs human all
Genomic context
Location: 9 A5.3; 9 29.76 cM See Crabp1 in Genome Data Viewer
Exon count: 4
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (54764748..54773110)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (54612615..54620916)
Chromosome 9 - NC_000075.6
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Transcript information: This gene has 1 transcript
Gene: Crabp1 ENSMUSG00000032291
Description cellular retinoic acid binding protein I [Source:MGI Symbol;Acc:MGI:88490] Gene Synonyms Crabp-1, CrabpI, Rbp-5 Location Chromosome 9: 54,764,748-54,773,110 forward strand. GRCm38:CM001002.2 About this gene This gene has 1 transcript (splice variant), 244 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Crabp1-201 ENSMUST00000034830.8 802 137aa ENSMUSP00000034830.8 Protein coding CCDS23195 P62965 TSL:1 GENCODE basic APPRIS P1
28.36 kb Forward strand 54.76Mb 54.77Mb 54.78Mb Genes (Comprehensive set... Crabp1-201 >protein coding
Contigs AC159892.2 > Regulatory Build
54.76Mb 54.77Mb 54.78Mb Reverse strand 28.36 kb
Regulation Legend Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
merged Ensembl/Havana
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Transcript: ENSMUST00000034830
8.36 kb Forward strand
Crabp1-201 >protein coding
ENSMUSP00000034... PDB-ENSP mappings Superfamily Calycin
Prints Cytosolic fatty-acid binding Pfam Lipocalin/cytosolic fatty-acid binding domain
PROSITE patterns Cytosolic fatty-acid binding
PANTHER Cellular retinoic acid-binding protein 1
Intracellular lipid binding protein Gene3D Calycin
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend
synonymous variant
Scale bar 0 20 40 60 80 100 137
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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