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Mouse Cab39 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cab39 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cab39 (NCBI Reference Sequence: NM_133781 ; Ensembl: ENSMUSG00000036707 ) is located on Mouse 1. 9 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000113360). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cab39 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-147F21 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:

Exon 3 starts from about 11.24% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 16809 bp, and the size of intron 4 for 3'-loxP site insertion: 4793 bp. The size of effective cKO region: ~2582 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 3 4 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cab39 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(9082bp) | A(26.6% 2416) | C(20.27% 1841) | T(32.18% 2923) | G(20.94% 1902)

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% chr1 + 85832074 85835073 3000 browser details YourSeq 117 179 2391 3000 93.4% chr1 - 71773221 71889050 115830 browser details YourSeq 110 185 2362 3000 94.4% chrX + 134633457 135019970 386514 browser details YourSeq 105 199 2400 3000 88.4% chr1 + 182053825 182198313 144489 browser details YourSeq 99 39 297 3000 87.2% chr1 - 82101154 82101406 253 browser details YourSeq 88 177 747 3000 81.2% chr17 - 34578795 34579318 524 browser details YourSeq 87 167 273 3000 94.9% chr6 - 128781314 128781422 109 browser details YourSeq 87 36 273 3000 94.1% chr1 - 180939950 180940269 320 browser details YourSeq 85 173 273 3000 96.8% chr9 + 37469471 37469573 103 browser details YourSeq 84 175 277 3000 95.7% chr19 + 6991373 6991477 105 browser details YourSeq 83 167 273 3000 87.7% chr10 - 80642318 80642423 106 browser details YourSeq 83 167 265 3000 96.7% chr7 + 129586444 129586544 101 browser details YourSeq 83 175 277 3000 94.7% chr14 + 5236839 5236943 105 browser details YourSeq 83 175 277 3000 94.7% chr14 + 5589444 5589548 105 browser details YourSeq 82 178 273 3000 96.7% chr8 + 102598734 102598831 98 browser details YourSeq 81 177 271 3000 97.8% chr14 - 47618759 47619315 557 browser details YourSeq 81 175 273 3000 95.6% chr11 - 23324655 23324755 101 browser details YourSeq 80 178 273 3000 96.6% chr10 - 77898980 77899077 98 browser details YourSeq 80 162 267 3000 91.8% chr1 + 50302191 50302298 108 browser details YourSeq 79 179 277 3000 92.1% chr2 - 130904936 130905032 97

Note: The 3000 bp section upstream of Exon 3 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% chr1 + 85837656 85840655 3000 browser details YourSeq 42 1402 1674 3000 95.7% chr10 + 67443228 67443591 364 browser details YourSeq 29 1659 1718 3000 94.0% chr14 - 60397921 60397982 62 browser details YourSeq 29 1661 1709 3000 82.3% chr7 + 67021956 67022005 50 browser details YourSeq 29 917 981 3000 96.8% chr17 + 37215564 37215630 67 browser details YourSeq 25 1425 1459 3000 85.8% chr5 + 33362043 33362077 35 browser details YourSeq 25 917 944 3000 96.5% chr1 + 171436409 171436437 29 browser details YourSeq 23 1661 1693 3000 84.9% chr10 - 119194969 119195001 33 browser details YourSeq 20 870 889 3000 100.0% chr17 - 5074156 5074175 20 browser details YourSeq 20 2864 2889 3000 88.5% chr14 + 11662306 11662331 26

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Cab39 calcium binding protein 39 [ Mus musculus (house mouse) ] Gene ID: 12283, updated on 24-Oct-2019

Gene summary

Official Symbol Cab39 provided by MGI Official Full Name calcium binding protein 39 provided by MGI Primary source MGI:MGI:107438 See related Ensembl:ENSMUSG00000036707 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 MO25; C78372; AA408805; AA960512; MO25alpha Expression Ubiquitous expression in bladder adult (RPKM 37.9), colon adult (RPKM 29.2) and 28 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 C5 See Cab39 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (85793441..85851577)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (87690022..87748152)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 5 transcripts

Gene: Cab39 ENSMUSG00000036707

Description calcium binding protein 39 [Source:MGI Symbol;Acc:MGI:107438] Gene Synonyms 39kDa, MO25alpha Location Chromosome 1: 85,793,441-85,851,576 forward strand. GRCm38:CM000994.2 About this gene This gene has 5 transcripts (splice variants), 204 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cab39-202 ENSMUST00000113360.7 3805 341aa ENSMUSP00000108987.1 Protein coding CCDS15112 Q06138 TSL:1 GENCODE basic APPRIS P1

Cab39-201 ENSMUST00000097666.3 2369 341aa ENSMUSP00000095270.2 Protein coding CCDS15112 Q06138 TSL:5 GENCODE basic APPRIS P1

Cab39-203 ENSMUST00000126962.2 580 38aa ENSMUSP00000116086.1 Protein coding - D3Z704 CDS 3' incomplete TSL:3

Cab39-204 ENSMUST00000130754.7 437 104aa ENSMUSP00000114690.1 Protein coding - D3YV52 CDS 3' incomplete TSL:3

Cab39-205 ENSMUST00000187623.1 2991 No protein - Retained intron - - TSL:NA

78.14 kb Forward strand 85.80Mb 85.82Mb 85.84Mb 85.86Mb (Comprehensive set... 9930111H07Rik-201 >lncRNA Cab39-205 >retained intron

Gm22906-201 >misc RCNaAb39-204 >protein coding

Cab39-202 >protein coding

Cab39-203 >protein coding

Cab39-201 >protein coding

Contigs < AC161342.10 < AC107707.10 Regulatory Build

85.80Mb 85.82Mb 85.84Mb 85.86Mb Reverse strand 78.14 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000113360

58.14 kb Forward strand

Cab39-202 >protein coding

ENSMUSP00000108... Superfamily Armadillo-type fold Pfam Mo25-like PANTHER PTHR10182:SF11

Mo25-like Gene3D Armadillo-like helical

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 341

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

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