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Mouse Jam3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Jam3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Jam3 gene (NCBI Reference Sequence: NM_023277 ; Ensembl: ENSMUSG00000031990 ) is located on Mouse chromosome 9. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000034472). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Jam3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-375B13 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: Approximately 60% of mice homozygous for a targeted mutation exhibit postnatal lethality. Males are infertile and display small testes and arrested differentiation of round spermatids into spermatozoa, as shown by the absence of acrosomes, elongated nuclei, and morphological signs of polarization.

Exon 4 starts from about 27.63% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 3490 bp, and the size of intron 5 for 3'-loxP site insertion: 1055 bp. The size of effective cKO region: ~1279 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 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Jam3 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(7779bp) | A(25.18% 1959) | C(21.67% 1686) | T(27.19% 2115) | G(25.95% 2019)

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 - 27102267 27105266 3000 browser details YourSeq 76 2050 2665 3000 93.2% chr15 - 99587555 99776426 188872 browser details YourSeq 74 1980 2091 3000 94.2% chr1 - 73546275 73546419 145 browser details YourSeq 71 2614 2734 3000 92.7% chr4 + 33678394 33678574 181 browser details YourSeq 70 2613 2780 3000 93.8% chr11 + 100923745 101219169 295425 browser details YourSeq 69 1987 2105 3000 85.8% chr13 - 98236176 98236310 135 browser details YourSeq 68 2633 2750 3000 92.6% chr7 - 6473648 6473822 175 browser details YourSeq 67 2613 2721 3000 95.9% chr8 - 25647117 25647299 183 browser details YourSeq 67 1984 2105 3000 87.0% chr6 - 88217687 88218076 390 browser details YourSeq 65 2005 2140 3000 86.6% chr9 - 71152616 71152756 141 browser details YourSeq 65 2633 2734 3000 93.5% chr11 - 6668144 6668321 178 browser details YourSeq 65 2613 2730 3000 93.3% chr9 + 65563396 65563562 167 browser details YourSeq 65 2613 2974 3000 94.6% chr11 + 78173436 78173922 487 browser details YourSeq 64 2052 2666 3000 91.9% chr15 - 38558861 38685147 126287 browser details YourSeq 64 2644 2730 3000 87.3% chr13 - 43627975 43628130 156 browser details YourSeq 63 1988 2084 3000 87.9% chr12 - 102575726 102575895 170 browser details YourSeq 62 2616 2721 3000 93.1% chr13 + 103979142 103979286 145 browser details YourSeq 61 2616 2725 3000 93.0% chr11 - 86035621 86035789 169 browser details YourSeq 60 2633 2734 3000 91.7% chr11 - 6783902 6784072 171 browser details YourSeq 60 2613 2720 3000 92.9% chr12 + 84659743 84659917 175

Note: The 3000 bp section upstream of Exon 4 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 - 27097988 27100987 3000 browser details YourSeq 120 167 342 3000 85.4% chr17 + 56523279 56523450 172 browser details YourSeq 116 167 339 3000 89.1% chr2 - 104709204 104709373 170 browser details YourSeq 116 167 344 3000 84.1% chr11 - 55138897 55139052 156 browser details YourSeq 115 167 344 3000 82.7% chr9 - 10672222 10672375 154 browser details YourSeq 114 187 345 3000 87.9% chr1 - 37257371 37257520 150 browser details YourSeq 112 185 344 3000 84.0% chr8 + 32550631 32550773 143 browser details YourSeq 111 186 331 3000 91.2% chr10 - 77615548 77615696 149 browser details YourSeq 111 167 342 3000 83.4% chr12 + 102207824 102207977 154 browser details YourSeq 110 167 321 3000 89.3% chr10 - 111784055 111784532 478 browser details YourSeq 108 165 319 3000 90.6% chr2 - 107004506 107004661 156 browser details YourSeq 108 167 337 3000 81.8% chr1 - 180655331 180655477 147 browser details YourSeq 107 187 341 3000 85.3% chr2 + 126976810 126976960 151 browser details YourSeq 105 185 344 3000 82.9% chr14 - 34401430 34401576 147 browser details YourSeq 105 188 319 3000 92.0% chr5 + 100354144 100354294 151 browser details YourSeq 105 186 333 3000 83.4% chr2 + 69728170 69728308 139 browser details YourSeq 105 185 344 3000 83.6% chr15 + 45708645 45708787 143 browser details YourSeq 105 197 441 3000 82.3% chr12 + 105085642 105085834 193 browser details YourSeq 104 207 344 3000 92.7% chr4 - 15390748 15390893 146 browser details YourSeq 104 186 328 3000 85.3% chr14 - 53292226 53292361 136

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Jam3 junction adhesion molecule 3 [ Mus musculus (house mouse) ] Gene ID: 83964, updated on 10-Oct-2019

Gene summary

Official Symbol Jam3 provided by MGI Official Full Name junction adhesion molecule 3 provided by MGI Primary source MGI:MGI:1933825 See related Ensembl:ENSMUSG00000031990 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 JAM-3; JAM-C; Jcam3; 1110002N23Rik Expression Broad expression in CNS E11.5 (RPKM 37.0), limb E14.5 (RPKM 30.4) and 21 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 A4 See Jam3 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (27097384..27155421, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (26904969..26962965, complement)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 5 transcripts

Gene: Jam3 ENSMUSG00000031990

Description junction adhesion molecule 3 [Source:MGI Symbol;Acc:MGI:1933825] Gene Synonyms 1110002N23Rik, JAM-3, JAM-C, Jcam3 Location Chromosome 9: 27,097,384-27,155,421 reverse strand. GRCm38:CM001002.2 About this gene This gene has 5 transcripts (splice variants), 255 orthologues, 17 paralogues, is a member of 1 Ensembl protein family and is associated with 39 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Jam3-201 ENSMUST00000034472.15 1942 310aa ENSMUSP00000034472.8 Protein coding CCDS22939 Q9D8B7 TSL:1 GENCODE basic APPRIS P1

Jam3-202 ENSMUST00000167074.1 1234 No protein - Retained intron - - TSL:1

Jam3-204 ENSMUST00000213682.1 597 No protein - Retained intron - - TSL:2

Jam3-205 ENSMUST00000215446.1 863 No protein - lncRNA - - TSL:3

Jam3-203 ENSMUST00000213170.1 731 No protein - lncRNA - - TSL:3

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78.04 kb Forward strand

27.10Mb 27.12Mb 27.14Mb 27.16Mb Genes Ncapd3-202 >protein codinGgm48796-201 >lncRNA (Comprehensive set...

Ncapd3-201 >protein coding

Ncapd3-205 >protein coding

Ncapd3-204 >lncRNA

Ncapd3-203 >lncRNA

Contigs CT025660.9 > Genes < Jam3-201protein coding (Comprehensive set...

< Jam3-205lncRNA

< Jam3-203lncRNA

< Jam3-202retained intron

< Jam3-204retained intron

Regulatory Build

27.10Mb 27.12Mb 27.14Mb 27.16Mb Reverse strand 78.04 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000034472

< Jam3-201protein coding

Reverse strand 58.01 kb

ENSMUSP00000034... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin V-set domain

Immunoglobulin subtype 2

Immunoglobulin subtype Pfam Immunoglobulin V-set domain PF13927

PROSITE profiles Immunoglobulin-like domain PANTHER PTHR44598:SF2

PTHR44598 Gene3D Immunoglobulin-like fold CDD cd00096

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 310

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

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