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Mouse Svil Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Svil (NCBI Reference Sequence: NM_153153 ; Ensembl: ENSMUSG00000024236 ) is located on Mouse 18. 35 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 35 (Transcript: ENSMUST00000025079). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Svil gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-193M5 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 knock-out allele exhibit enhanched adhesion and thrombus formation.

Exon 3 starts from about 2.47% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1969 bp, and the size of intron 3 for 3'-loxP site insertion: 4523 bp. The size of effective cKO region: ~1110 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 2 3 35 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Svil 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(7610bp) | A(26.4% 2009) | C(24.38% 1855) | T(27.61% 2101) | G(21.62% 1645)

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% chr18 + 5045636 5048635 3000 browser details YourSeq 60 2389 2549 3000 86.6% chr1 + 84859233 84859389 157 browser details YourSeq 48 2402 2552 3000 72.3% chr14 - 12103346 12103448 103 browser details YourSeq 42 2442 2549 3000 95.7% chr1 + 84859136 84859287 152 browser details YourSeq 34 1529 1615 3000 85.5% chr9 - 60497686 60497773 88 browser details YourSeq 34 2389 2457 3000 73.0% chr1 + 84859131 84859185 55 browser details YourSeq 32 1526 1610 3000 97.1% chr4 + 19913584 19913668 85 browser details YourSeq 29 2367 2502 3000 51.7% chr14 - 55088706 55088751 46 browser details YourSeq 28 2489 2549 3000 70.0% chr1 + 84859137 84859185 49 browser details YourSeq 27 2369 2412 3000 96.6% chr17 + 29725810 29725854 45 browser details YourSeq 27 2369 2412 3000 89.7% chr16 + 3745261 3745303 43 browser details YourSeq 23 443 467 3000 87.5% chr1 + 122169649 122169672 24

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% chr18 + 5049746 5052745 3000 browser details YourSeq 210 600 925 3000 87.8% chr1 + 50872541 50976342 103802 browser details YourSeq 209 592 985 3000 88.0% chr1 - 106919567 106920220 654 browser details YourSeq 202 592 925 3000 88.5% chr14 + 39396781 39397123 343 browser details YourSeq 198 592 1363 3000 78.2% chr4 + 99835789 99836483 695 browser details YourSeq 194 592 925 3000 87.6% chr18 - 36212402 36212738 337 browser details YourSeq 193 592 925 3000 86.2% chr13 - 85048238 85048561 324 browser details YourSeq 192 592 925 3000 88.1% chr15 + 13510864 13511195 332 browser details YourSeq 190 611 925 3000 89.7% chr14 - 97221076 97436885 215810 browser details YourSeq 187 600 925 3000 84.6% chrX + 68616046 68616377 332 browser details YourSeq 186 632 925 3000 89.1% chrX - 75055086 75055379 294 browser details YourSeq 186 600 931 3000 87.6% chr2 - 78223977 78224306 330 browser details YourSeq 185 576 925 3000 86.1% chr18 - 7851874 7852187 314 browser details YourSeq 183 601 925 3000 82.1% chr19 + 11267585 11267908 324 browser details YourSeq 182 600 901 3000 86.4% chr1 + 108915344 108915645 302 browser details YourSeq 182 600 904 3000 88.4% chr1 + 83552704 83553005 302 browser details YourSeq 181 592 922 3000 88.7% chr19 + 13881357 13881683 327 browser details YourSeq 180 592 925 3000 84.6% chr3 - 17690724 17691051 328 browser details YourSeq 179 588 925 3000 85.4% chr6 + 134339755 134340092 338 browser details YourSeq 177 592 925 3000 76.7% chr6 + 55957661 55957997 337

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Svil supervillin [ Mus musculus (house mouse) ] Gene ID: 225115, updated on 10-Sep-2019

Gene summary

Official Symbol Svil provided by MGI Official Full Name supervillin provided by MGI Primary source MGI:MGI:2147319 See related Ensembl:ENSMUSG00000024236 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 AU024053; B430302E16Rik Expression Broad expression in bladder adult (RPKM 33.3), heart adult (RPKM 16.5) and 20 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 A1 See Svil in Genome Data Viewer

Exon count: 44

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (4920467..5119293)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (5046587..5119291)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 15 transcripts

Gene: Svil ENSMUSG00000024236

Description supervillin [Source:MGI Symbol;Acc:MGI:2147319] Gene Synonyms B430302E16Rik Location Chromosome 18: 4,920,540-5,119,299 forward strand. GRCm38:CM001011.2 About this gene This gene has 15 transcripts (splice variants), 223 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Svil- ENSMUST00000126977.7 7907 2170aa ENSMUSP00000115078.1 Protein coding CCDS37720 Q8K4L3 TSL:5 203 GENCODE basic APPRIS P3

Svil- ENSMUST00000025079.15 7433 2170aa ENSMUSP00000025079.9 Protein coding CCDS37720 Q8K4L3 TSL:1 201 GENCODE basic APPRIS P3

Svil- ENSMUST00000140448.7 7423 2170aa ENSMUSP00000119803.1 Protein coding CCDS37720 Q8K4L3 TSL:5 210 GENCODE basic APPRIS P3

Svil- ENSMUST00000143254.7 6594 1766aa ENSMUSP00000119287.1 Protein coding CCDS84352 Q8K4L3 TSL:5 211 GENCODE basic APPRIS ALT2

Svil- ENSMUST00000210707.1 7633 2257aa ENSMUSP00000147843.1 Protein coding - A0A1B0GS91 TSL:5 215 GENCODE basic APPRIS ALT2

Svil- ENSMUST00000127297.7 6243 2056aa ENSMUSP00000115223.1 Protein coding - E9Q3Z5 TSL:5 204 GENCODE basic APPRIS ALT2

Svil- ENSMUST00000146723.1 507 169aa ENSMUSP00000115591.1 Protein coding - F6TBK9 CDS 5' and 3' 212 incomplete TSL:3

Svil- ENSMUST00000153016.7 497 40aa ENSMUSP00000121497.1 Protein coding - D3Z2X9 CDS 3' 214 incomplete TSL:2

Svil- ENSMUST00000131609.7 6420 2031aa ENSMUSP00000122242.1 Nonsense mediated - Q8K4L2 TSL:5 207 decay

Svil- ENSMUST00000125512.7 4060 749aa ENSMUSP00000121972.1 Nonsense mediated - F6R6A4 CDS 5' 202 decay incomplete TSL:5

Svil- ENSMUST00000129543.1 1732 No - Retained intron - - TSL:2 205 protein

Svil- ENSMUST00000131210.7 1560 No - Retained intron - - TSL:1 206 protein

Svil- ENSMUST00000138258.7 1430 No - Retained intron - - TSL:5 208 protein

Svil- ENSMUST00000139761.1 523 No - Retained intron - - TSL:2 209 protein

Svil- ENSMUST00000148564.1 1218 No - lncRNA - - TSL:1 213 protein

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218.76 kb Forward strand

4.95Mb 5.00Mb 5.05Mb 5.10Mb (Comprehensive set... Svil-203 >protein coding

Svil-206 >retained intron Svil-202 >nonsense mediated decay

Svil-208 >retained intron Svil-209 >retained intron

Svil-211 >protein coding

Svil-214 >protein coding Svil-205 >retained intron

Svil-210 >protein coding

Svil-213 >lncRNA Svil-215 >protein coding

Svil-204 >protein coding

Svil-207 >nonsense mediated decay

Svil-201 >protein coding

Svil-212 >protein coding

Contigs AC124770.4 > < AC115928.10 Regulatory Build

4.95Mb 5.00Mb 5.05Mb 5.10Mb Reverse strand 218.76 kb

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

Gene Legend Protein Coding

Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000025079

72.71 kb Forward strand

Svil-201 >protein coding

ENSMUSP00000025... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF55753 headpiece domain superfamily SMART Villin/ Villin headpiece Prints Villin/Gelsolin Pfam Gelsolin-like domain Villin headpiece

PROSITE profiles Villin headpiece PANTHER Villin/Gelsolin

Supervillin Gene3D ADF-H/Gelsolin-like domain superfamily Villin headpiece domain superfamily CDD cd11289 cd11293

cd11280 cd11288

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

Variant Legend inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2170

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

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