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Mouse Cadm3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cadm3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cadm3 gene (NCBI Reference Sequence: NM_053199 ; Ensembl: ENSMUSG00000005338 ) is located on Mouse chromosome 1. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000111220). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cadm3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-341H3 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 null allele exhibit delayed myelination. Other mice with ubiquitous conditional deletion of the gene do not display neurological abnormalities.

Exon 6 starts from about 57.74% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 2560 bp, and the size of intron 6 for 3'-loxP site insertion: 445 bp. The size of effective cKO region: ~563 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 6 7 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cadm3 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. 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(7035bp) | A(27.12% 1908) | C(22.15% 1558) | T(27.25% 1917) | G(23.48% 1652)

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 - 173342010 173345009 3000 browser details YourSeq 159 158 2986 3000 94.0% chr13 + 7966445 8235303 268859 browser details YourSeq 121 167 483 3000 86.7% chr10 - 107609686 107609969 284 browser details YourSeq 117 158 484 3000 86.4% chr7 - 4672706 4672959 254 browser details YourSeq 117 161 488 3000 97.7% chr11 + 36017015 36017451 437 browser details YourSeq 114 158 483 3000 83.2% chr15 + 5279754 5279892 139 browser details YourSeq 113 2878 3000 3000 96.0% chr10 + 74473704 74473826 123 browser details YourSeq 110 2879 3000 3000 95.1% chr9 - 119693636 119693757 122 browser details YourSeq 110 2875 3000 3000 93.7% chr12 + 17022096 17022221 126 browser details YourSeq 109 2878 3000 3000 94.4% chr8 - 15163181 15163303 123 browser details YourSeq 109 2878 3000 3000 94.4% chr10 - 62860002 62860124 123 browser details YourSeq 109 2878 3000 3000 94.4% chr1 - 132789426 132789548 123 browser details YourSeq 108 2879 3000 3000 94.3% chr11 - 23331027 23331148 122 browser details YourSeq 107 2878 3000 3000 93.5% chr9 - 14491444 14491566 123 browser details YourSeq 107 2878 3000 3000 93.5% chr8 - 15483784 15483906 123 browser details YourSeq 107 2882 3000 3000 95.0% chr15 - 23495931 23496049 119 browser details YourSeq 107 2859 3000 3000 86.7% chr13 + 54591347 54591478 132 browser details YourSeq 107 2862 3000 3000 92.2% chr1 + 48675769 48676041 273 browser details YourSeq 106 2877 3000 3000 92.8% chrX - 166299726 166299849 124 browser details YourSeq 106 2877 3000 3000 92.8% chr13 - 84717681 84717804 124

Note: The 3000 bp section upstream of Exon 6 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 - 173338447 173341446 3000 browser details YourSeq 48 1556 1630 3000 81.9% chr14 + 92422371 92422439 69 browser details YourSeq 41 1534 1624 3000 83.7% chr13 - 98669669 98669756 88 browser details YourSeq 39 1584 1630 3000 91.5% chr14 - 106330368 106330414 47 browser details YourSeq 39 1557 1624 3000 83.8% chr12 - 56125810 56125872 63 browser details YourSeq 39 1548 1593 3000 86.4% chr15 + 43696512 43696555 44 browser details YourSeq 38 1582 1630 3000 90.5% chr9 - 32249361 32249408 48 browser details YourSeq 37 1578 1630 3000 85.0% chr6 - 123749195 123749247 53 browser details YourSeq 37 1546 1596 3000 86.3% chrX + 82992514 82992564 51 browser details YourSeq 37 1584 1630 3000 89.4% chr13 + 20276743 20276789 47 browser details YourSeq 35 1582 1630 3000 85.8% chr14 + 20572236 20572284 49 browser details YourSeq 34 1553 1596 3000 88.7% chr10 + 101166530 101166573 44 browser details YourSeq 33 1579 1624 3000 91.5% chr5 + 25572818 25572862 45 browser details YourSeq 32 1137 1182 3000 91.9% chr5 + 136828530 136828576 47 browser details YourSeq 31 1578 1618 3000 87.9% chr11 - 110353730 110353770 41 browser details YourSeq 29 1585 1630 3000 94.0% chr12 - 44666636 44666681 46 browser details YourSeq 27 1585 1617 3000 84.4% chr10 - 17207202 17207233 32 browser details YourSeq 24 1141 1168 3000 85.2% chr5 + 139054795 139054821 27 browser details YourSeq 22 1137 1168 3000 84.4% chr18 - 84778156 84778187 32 browser details YourSeq 20 465 502 3000 76.4% chr4 - 139786180 139786217 38

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cadm3 cell adhesion molecule 3 [ Mus musculus (house mouse) ] Gene ID: 94332, updated on 11-Sep-2019

Gene summary

Official Symbol Cadm3 provided by MGI Official Full Name cell adhesion molecule 3 provided by MGI Primary source MGI:MGI:2137858 See related Ensembl:ENSMUSG00000005338 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 BIgR; Necl1; Tsll1; Igsf4b; Necl-1; SynCAM3 Expression Biased expression in cerebellum adult (RPKM 149.0), frontal lobe adult (RPKM 64.2) and 7 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 H3 See Cadm3 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (173331892..173368139, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (175264385..175297826, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 5 transcripts

Gene: Cadm3 ENSMUSG00000005338

Description cell adhesion molecule 3 [Source:MGI Symbol;Acc:MGI:2137858] Gene Synonyms BIgR, Igsf4b, Necl-1, Necl1, SynCAM3, Tsll1 Location Chromosome 1: 173,333,258-173,367,695 reverse strand. GRCm38:CM000994.2 About this gene This gene has 5 transcripts (splice variants), 195 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

Cadm3-202 ENSMUST00000111220.7 5155 396aa ENSMUSP00000106851.1 Protein coding CCDS15528 Q99N28 TSL:1 GENCODE basic APPRIS P2

Cadm3-201 ENSMUST00000005470.4 2518 430aa ENSMUSP00000005470.4 Protein coding - K4DI58 TSL:5 GENCODE basic APPRIS ALT1

Cadm3-205 ENSMUST00000157032.7 2746 No protein - Retained intron - - TSL:1

Cadm3-204 ENSMUST00000136540.1 855 No protein - Retained intron - - TSL:1

Cadm3-203 ENSMUST00000126963.1 774 No protein - Retained intron - - TSL:3

54.44 kb Forward strand 173.33Mb 173.34Mb 173.35Mb 173.36Mb 173.37Mb Genes Aim2-202 >protein coding (Comprehensive set...

Contigs < AC087781.17 AC084073.22 > Genes (Comprehensive set... < Ackr1-203lncRNA < Cadm3-205retained intron

< Ackr1-201protein coding < Cadm3-203retained intron

< Ackr1-202protein coding

< Cadm3-202protein coding

< Cadm3-201protein coding

< Cadm3-204retained intron

Regulatory Build

173.33Mb 173.34Mb 173.35Mb 173.36Mb 173.37Mb Reverse strand 54.44 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000111220

< Cadm3-202protein coding

Reverse strand 34.44 kb

ENSMUSP00000106... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin subtype 2 Neurexin/syndecan/glycophorin C

Immunoglobulin subtype Pfam Immunoglobulin I-set CD80-like, immunoglobulin C2-set

PF13927 PROSITE profiles Immunoglobulin-like domain PANTHER PTHR45889

PTHR45889:SF5 Gene3D Immunoglobulin-like fold CDD cd05882 cd07705

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

Variant Legend splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 396

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

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