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Mouse Scin Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Scin (NCBI Reference Sequence: NM_001146196 ; Ensembl: ENSMUSG00000002565 ) is located on Mouse 12. 16 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 16 (Transcript: ENSMUST00000002640). 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 Scin gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-76H11 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 conditional allele knocked-out in osteoclasts exhibit impaired osteoclast differentiation and reduced peridontal disease-mediated bone loss.

Exon 2 starts from about 9.32% 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: 5874 bp, and the size of intron 2 for 3'-loxP site insertion: 3152 bp. The size of effective cKO region: ~655 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 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Scin 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(7155bp) | A(29.07% 2080) | C(20.8% 1488) | T(30.16% 2158) | G(19.97% 1429)

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% chr12 - 40128286 40131285 3000 browser details YourSeq 86 2789 2965 3000 78.6% chr13 - 118290721 118290888 168 browser details YourSeq 80 1572 1737 3000 85.0% chr2 + 144413733 144413902 170 browser details YourSeq 68 1586 1692 3000 82.3% chr6 - 82299551 82299658 108 browser details YourSeq 66 1592 1696 3000 79.7% chr4 - 134085203 134085306 104 browser details YourSeq 66 1581 1683 3000 85.2% chr17 - 72517029 72517371 343 browser details YourSeq 66 1586 1690 3000 87.7% chr11 - 82642348 82642630 283 browser details YourSeq 65 1594 1695 3000 84.3% chr9 + 115225299 115225401 103 browser details YourSeq 65 1590 1772 3000 84.1% chr6 + 34795488 34795706 219 browser details YourSeq 63 1587 1677 3000 84.7% chr7 - 98666539 98666629 91 browser details YourSeq 62 1589 1692 3000 79.8% chr1 + 131983414 131983516 103 browser details YourSeq 61 1593 1692 3000 81.0% chr1 - 93105234 93105334 101 browser details YourSeq 61 1585 1692 3000 78.8% chr1 + 85041454 85041562 109 browser details YourSeq 60 1582 1696 3000 80.7% chr17 + 11934291 11934409 119 browser details YourSeq 60 1613 1692 3000 89.8% chr11 + 21973197 22048139 74943 browser details YourSeq 60 1585 1677 3000 82.7% chr1 + 125574120 125574212 93 browser details YourSeq 59 1605 1722 3000 91.7% chr4 - 105487370 105487488 119 browser details YourSeq 59 1589 1692 3000 83.6% chr1 - 40288354 40288455 102 browser details YourSeq 57 1587 1696 3000 78.3% chr7 + 36599582 36599694 113 browser details YourSeq 56 1585 1693 3000 83.6% chr5 - 127040641 127040748 108

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% chr12 - 40124631 40127630 3000 browser details YourSeq 336 1840 2241 3000 92.3% chr10 + 111124483 111357779 233297 browser details YourSeq 334 1844 2241 3000 91.4% chr13 - 50451585 50451980 396 browser details YourSeq 332 1841 2241 3000 91.8% chr18 - 46538134 46538534 401 browser details YourSeq 332 1840 2239 3000 91.9% chr10 - 119036349 119036747 399 browser details YourSeq 332 1839 2251 3000 91.1% chrX + 12505070 12505491 422 browser details YourSeq 332 1845 2241 3000 92.0% chr9 + 43360949 43361346 398 browser details YourSeq 330 1841 2241 3000 91.3% chr9 + 40843097 40843498 402 browser details YourSeq 330 1841 2241 3000 92.1% chr8 + 14640248 14724425 84178 browser details YourSeq 329 1838 2244 3000 91.0% chr6 + 29123518 29123923 406 browser details YourSeq 329 1841 2241 3000 91.1% chr5 + 102097173 102097573 401 browser details YourSeq 329 1841 2242 3000 91.1% chr15 + 85225262 85225660 399 browser details YourSeq 329 1840 2241 3000 92.1% chr15 + 75590847 75591250 404 browser details YourSeq 327 1841 2242 3000 89.7% chr5 - 150393008 150393405 398 browser details YourSeq 327 1841 2242 3000 89.7% chr5 - 148761083 148761480 398 browser details YourSeq 327 1840 2241 3000 90.7% chr10 - 59381318 59381717 400 browser details YourSeq 327 1841 2235 3000 91.7% chr5 + 151292761 151293152 392 browser details YourSeq 327 1646 2241 3000 90.2% chr5 + 90797896 90798579 684 browser details YourSeq 326 1840 2243 3000 90.0% chr5 - 148378443 148378844 402 browser details YourSeq 326 1841 2242 3000 90.1% chr13 - 53036220 53036620 401

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 information: Scin scinderin [ Mus musculus (house mouse) ] Gene ID: 20259, updated on 12-Aug-2019

Gene summary

Official Symbol Scin provided by MGI Official Full Name scinderin provided by MGI Primary source MGI:MGI:1306794 See related Ensembl:ENSMUSG00000002565 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 AW545522; adseverin Expression Biased expression in colon adult (RPKM 56.3), large intestine adult (RPKM 22.6) and 4 other tissuesS ee more Orthologs human all

Genomic context

Location: 12; 12 B1 See Scin in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (40059769..40134228, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (40786356..40860815, complement)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 2 transcripts

Gene: Scin ENSMUSG00000002565

Description scinderin [Source:MGI Symbol;Acc:MGI:1306794] Gene Synonyms adseverin Location Chromosome 12: 40,059,769-40,134,228 reverse strand. GRCm38:CM001005.2 About this gene This gene has 2 transcripts (splice variants), 171 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Scin-201 ENSMUST00000002640.5 2995 715aa ENSMUSP00000002640.5 Protein coding CCDS49055 Q60604 TSL:1 GENCODE basic APPRIS P1

Scin-202 ENSMUST00000078481.13 2654 615aa ENSMUSP00000077573.7 Protein coding CCDS25891 Q60604 TSL:1 GENCODE basic

94.46 kb Forward strand 40.06Mb 40.08Mb 40.10Mb 40.12Mb 40.14Mb Contigs < AC131921.9 < AC174598.2 Genes (Comprehensive set... < Scin-202protein coding

< Scin-201protein coding

Regulatory Build

40.06Mb 40.08Mb 40.10Mb 40.12Mb 40.14Mb Reverse strand 94.46 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

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Transcript: ENSMUST00000002640

< Scin-201protein coding

Reverse strand 74.46 kb

ENSMUSP00000002... Low complexity (Seg) Superfamily SSF55753

Gelsolin-like domain superfamily SMART Villin/ Prints Villin/Gelsolin Pfam Gelsolin-like domain PANTHER Villin/Gelsolin

Adseverin Gene3D ADF-H/Gelsolin-like domain superfamily CDD cd11290 cd11289 cd11292 cd11293 cd11288 cd11291

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

Variant Legend frameshift variant missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 715

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

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