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Mouse Scamp3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Scamp3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Scamp3 gene (NCBI Reference Sequence: NM_011886 ; Ensembl: ENSMUSG00000028049 ) is located on Mouse chromosome 3. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000029684). 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 Scamp3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-214B5 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 13.85% 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: 1027 bp, and the size of intron 4 for 3'-loxP site insertion: 814 bp. The size of effective cKO region: ~846 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'

13 1 2 3 4 5 6 7 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Clk2 Homology arm Exon of mouse Scamp3 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(7343bp) | A(23.87% 1753) | C(23.27% 1709) | T(25.6% 1880) | G(27.25% 2001)

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% chr3 + 89175846 89178845 3000 browser details YourSeq 48 2533 2646 3000 87.5% chr6 - 120991331 120991442 112 browser details YourSeq 45 379 488 3000 94.2% chr11 + 70672934 70673044 111 browser details YourSeq 44 379 497 3000 92.4% chr18 + 53761387 53761753 367 browser details YourSeq 40 2626 2731 3000 95.5% chr18 - 89843380 89843501 122 browser details YourSeq 40 2598 2657 3000 84.5% chr1 + 69452100 69452167 68 browser details YourSeq 39 2539 2655 3000 87.3% chr3 - 27212521 27212636 116 browser details YourSeq 37 443 491 3000 87.8% chr11 - 96879787 96879835 49 browser details YourSeq 37 2626 2731 3000 93.1% chr10 - 77639498 77639630 133 browser details YourSeq 35 2609 2657 3000 85.8% chr10 + 41512875 41512923 49 browser details YourSeq 34 446 489 3000 88.7% chr16 + 22292775 22292818 44 browser details YourSeq 34 2626 2729 3000 92.5% chr15 + 97835425 97835530 106 browser details YourSeq 34 373 482 3000 86.5% chr11 + 46071635 46071742 108 browser details YourSeq 31 2607 2655 3000 81.7% chr2 + 173285300 173285348 49 browser details YourSeq 30 2624 2655 3000 96.9% chr2 - 155273414 155273445 32 browser details YourSeq 30 407 477 3000 84.1% chr19 - 44690016 44690103 88 browser details YourSeq 30 446 488 3000 94.3% chr17 - 30569708 30569751 44 browser details YourSeq 30 2860 2903 3000 73.6% chr16 - 62378248 62378283 36 browser details YourSeq 30 2532 2561 3000 100.0% chr13 - 56672640 56672669 30 browser details YourSeq 30 373 483 3000 96.9% chr11 - 107754906 107755016 111

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% chr3 + 89179689 89182688 3000 browser details YourSeq 138 1834 2011 3000 92.6% chrX - 96525921 96526104 184 browser details YourSeq 137 1838 2011 3000 91.2% chr4 - 92525883 92526054 172 browser details YourSeq 134 1850 2010 3000 89.7% chr5 - 22084301 22084456 156 browser details YourSeq 132 1838 2005 3000 88.1% chr11 - 70503361 70503516 156 browser details YourSeq 132 1850 2011 3000 88.4% chr14 + 56709589 56709744 156 browser details YourSeq 132 1849 2010 3000 88.8% chr10 + 62819699 62819850 152 browser details YourSeq 131 1834 2014 3000 86.7% chr11 - 101455901 101456072 172 browser details YourSeq 130 1840 2005 3000 94.6% chr11 - 100729818 100729983 166 browser details YourSeq 129 1850 2005 3000 92.8% chr18 - 62460681 62544017 83337 browser details YourSeq 129 1848 2010 3000 87.1% chr16 - 33789455 33789611 157 browser details YourSeq 129 1864 2015 3000 93.9% chr14 - 45092777 45092933 157 browser details YourSeq 129 1850 2010 3000 87.5% chr6 + 71835266 71835416 151 browser details YourSeq 129 1850 2000 3000 89.7% chr17 + 25954481 25954625 145 browser details YourSeq 128 1847 2010 3000 93.3% chr4 - 116978769 116978943 175 browser details YourSeq 128 1850 2011 3000 91.0% chr17 - 46811039 46811199 161 browser details YourSeq 128 1850 2009 3000 87.1% chr4 + 107377296 107377449 154 browser details YourSeq 128 1850 2005 3000 88.0% chr16 + 94474162 94474311 150 browser details YourSeq 127 1864 2011 3000 93.1% chr17 - 63694255 63694401 147 browser details YourSeq 127 1852 2011 3000 89.4% chr9 + 86505015 86505171 157

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 protein information: Scamp3 secretory carrier membrane protein 3 [ Mus musculus (house mouse) ] Gene ID: 24045, updated on 12-Aug-2019

Gene summary

Official Symbol Scamp3 provided by MGI Official Full Name secretory carrier membrane protein 3 provided by MGI Primary source MGI:MGI:1346346 See related Ensembl:ENSMUSG00000028049 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 Sc3 Expression Ubiquitous expression in adrenal adult (RPKM 100.4), thymus adult (RPKM 60.9) and 28 other tissues See more

Genomic context

Location: 3; 3 F1 See Scamp3 in Genome Data Viewer Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (89177391..89182770)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (88981407..88986692)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 8 transcripts

Gene: Scamp3 ENSMUSG00000028049

Description secretory carrier membrane protein 3 [Source:MGI Symbol;Acc:MGI:1346346] Gene Synonyms Sc3 Location Chromosome 3: 89,177,473-89,182,765 forward strand. GRCm38:CM000996.2 About this gene This gene has 8 transcripts (splice variants), 189 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Scamp3-201 ENSMUST00000029684.14 1489 349aa ENSMUSP00000029684.8 Protein coding CCDS50959 O35609 TSL:1 GENCODE basic APPRIS P3

Scamp3-203 ENSMUST00000120697.7 1475 350aa ENSMUSP00000112846.1 Protein coding CCDS84640 Q3UXS0 TSL:1 GENCODE basic APPRIS ALT2

Scamp3-202 ENSMUST00000098941.4 1338 315aa ENSMUSP00000096540.4 Protein coding CCDS84641 E9Q855 TSL:1 GENCODE basic

Scamp3-205 ENSMUST00000129294.7 969 No protein - Retained intron - - TSL:5

Scamp3-208 ENSMUST00000156447.7 802 No protein - Retained intron - - TSL:3

Scamp3-206 ENSMUST00000136165.7 721 No protein - Retained intron - - TSL:2

Scamp3-204 ENSMUST00000124291.1 694 No protein - Retained intron - - TSL:2

Scamp3-207 ENSMUST00000148881.7 676 No protein - Retained intron - - TSL:2

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25.29 kb Forward strand 89.17Mb 89.18Mb 89.19Mb Genes (Comprehensive set... Clk2-202 >protein coding Scamp3-201 >protein coding Fam189b-208 >protein coding Fam189b-207 >retained intron

Clk2-201 >protein coding Scamp3-203 >protein coding Fam189b-201 >protein coding

Clk2-207 >retained intron Scamp3-206 >retained intron Fam189b-209 >nonsense mediated decay

Clk2-209 >protein coding Clk2-205 >retained intron Scamp3-202 >protein coding Fam189b-206 >nonsense mediated decay

Clk2-203 >protein coding Scamp3-208 >retained intron Fam189b-202 >protein coding

Clk2-204 >nonsense mediated decay Scamp3-207 >retained intron Fam189b-203 >protein coding

Clk2-206 >retained intron Clk2-211 >retained intron Scamp3-205 >retained intron Fam189b-205 >retained intFroanm189b-204 >retained intron

Clk2-208 >lncRNA Scamp3-204 >retained intron Fam189b-210 >lncRNA

Contigs AC161600.6 >

Genes < Gm16069-201lncRNA (Comprehensive set...

Regulatory Build

89.17Mb 89.18Mb 89.19Mb Reverse strand 25.29 kb

Regulation Legend

CTCF 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: ENSMUST00000029684

5.29 kb Forward strand

Scamp3-201 >protein coding

ENSMUSP00000029... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam SCAMP PANTHER PTHR10687:SF6

SCAMP

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 349

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

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