https://www.alphaknockout.com

Mouse Arhgap25 Knockout Project (CRISPR/Cas9)

Objective: To create a Arhgap25 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Arhgap25 gene (NCBI Reference Sequence: NM_001037727 ; Ensembl: ENSMUSG00000030047 ) is located on Mouse chromosome 6. 11 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000113637). Exon 3~4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit altered leukocyte transendothelial migration.

Exon 3 starts from about 13.48% of the coding region. Exon 3~4 covers 10.55% of the coding region. The size of effective KO region: ~4074 bp. The KO 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 3 4 11

Legends Exon of mouse Arhgap25 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 4 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(31.05% 621) | C(19.6% 392) | T(28.3% 566) | G(21.05% 421)

Note: The 2000 bp section upstream of Exon 3 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(26.9% 538) | C(22.45% 449) | T(31.45% 629) | G(19.2% 384)

Note: The 2000 bp section downstream of Exon 4 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 2000 1 2000 2000 100.0% chr6 - 87492266 87494265 2000 browser details YourSeq 56 1437 1543 2000 91.2% chr11 - 22476141 22476267 127 browser details YourSeq 49 1497 1574 2000 88.9% chr9 - 71827723 71827808 86 browser details YourSeq 48 1510 1586 2000 86.4% chr2 - 71768734 71768812 79 browser details YourSeq 47 1453 1557 2000 94.5% chr12 + 108869749 108870180 432 browser details YourSeq 46 1386 1468 2000 91.1% chr9 - 89670527 89670612 86 browser details YourSeq 45 1385 1556 2000 92.5% chr9 + 56921258 56921462 205 browser details YourSeq 44 1415 1468 2000 90.8% chr17 - 27068901 27068954 54 browser details YourSeq 44 1384 1556 2000 90.8% chr7 + 113541957 113542146 190 browser details YourSeq 43 1387 1555 2000 87.8% chr12 - 7612274 7612440 167 browser details YourSeq 43 1386 1476 2000 86.5% chr13 + 51533091 51533189 99 browser details YourSeq 43 1511 1575 2000 79.3% chr10 + 75765364 75765422 59 browser details YourSeq 42 1383 1439 2000 87.5% chr12 - 110416505 110416566 62 browser details YourSeq 42 1497 1574 2000 76.7% chr14 + 46301465 46301538 74 browser details YourSeq 41 1437 1554 2000 97.8% chr5 - 25772235 25772352 118 browser details YourSeq 41 1512 1574 2000 77.6% chr12 - 87052146 87052198 53 browser details YourSeq 40 1394 1468 2000 88.5% chr1 + 86067467 86067546 80 browser details YourSeq 39 1454 1556 2000 86.7% chr8 + 84540216 84540317 102 browser details YourSeq 38 1511 1556 2000 91.4% chr12 - 84644491 84644536 46 browser details YourSeq 38 1513 1562 2000 89.8% chr14 + 21366645 21366702 58

Note: The 2000 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 2000 1 2000 2000 100.0% chr6 - 87486192 87488191 2000 browser details YourSeq 59 1036 1195 2000 85.6% chr1 + 182594826 182594980 155 browser details YourSeq 48 1014 1195 2000 94.6% chr15 + 102868772 102868993 222 browser details YourSeq 44 1067 1182 2000 94.0% chr15 - 84652311 84652429 119 browser details YourSeq 43 1040 1097 2000 92.2% chr16 - 69018875 69019087 213 browser details YourSeq 42 1157 1601 2000 92.0% chr1 + 89060814 89061329 516 browser details YourSeq 40 1076 1210 2000 76.6% chr12 + 70151915 70152041 127 browser details YourSeq 37 1069 1208 2000 80.0% chr11 + 77210963 77211096 134 browser details YourSeq 34 1069 1170 2000 88.7% chr11 + 78335750 78335853 104 browser details YourSeq 30 1076 1191 2000 94.2% chr16 - 33092345 33092462 118 browser details YourSeq 29 1157 1195 2000 84.9% chr16 - 31723162 31723198 37 browser details YourSeq 29 1157 1194 2000 84.9% chr2 + 70653659 70653694 36 browser details YourSeq 29 1157 1195 2000 94.0% chr2 + 52874826 52874865 40 browser details YourSeq 29 1067 1097 2000 96.8% chr19 + 46980501 46980531 31 browser details YourSeq 28 1157 1194 2000 93.8% chr10 + 62917274 62917312 39 browser details YourSeq 27 1157 1195 2000 93.6% chr1 - 21613692 21613731 40 browser details YourSeq 26 1066 1093 2000 96.5% chr11 - 61846755 61846782 28 browser details YourSeq 25 1157 1189 2000 96.3% chr13 - 50481525 50481558 34 browser details YourSeq 24 1870 1898 2000 84.7% chr10 + 127140210 127140236 27 browser details YourSeq 24 1157 1188 2000 96.2% chr1 + 157239851 157239883 33

Note: The 2000 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: Arhgap25 Rho GTPase activating protein 25 [ Mus musculus (house mouse) ] Gene ID: 232201, updated on 12-Aug-2019

Gene summary

Official Symbol Arhgap25 provided by MGI Official Full Name Rho GTPase activating protein 25 provided by MGI Primary source MGI:MGI:2443687 See related Ensembl:ENSMUSG00000030047 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 A130039I20Rik Expression Broad expression in spleen adult (RPKM 4.7), thymus adult (RPKM 2.9) and 17 other tissues See more Orthologs human all

Genomic context

Location: 6; 6 D1 See Arhgap25 in Genome Data Viewer Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (87458544..87533261, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (87409379..87483229, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 5 transcripts

Gene: Arhgap25 ENSMUSG00000030047

Description Rho GTPase activating protein 25 [Source:MGI Symbol;Acc:MGI:2443687] Gene Synonyms A130039I20Rik Location Chromosome 6: 87,458,545-87,533,259 reverse strand. GRCm38:CM000999.2 About this gene This gene has 5 transcripts (splice variants), 197 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 15 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Arhgap25-203 ENSMUST00000113637.8 3671 648aa ENSMUSP00000109267.2 Protein coding CCDS20321 Q8BYW1 TSL:1 GENCODE basic APPRIS P4

Arhgap25-201 ENSMUST00000071024.6 2635 559aa ENSMUSP00000068964.6 Protein coding CCDS71790 Q8BYW1 TSL:1 GENCODE basic

Arhgap25-202 ENSMUST00000101197.8 2493 622aa ENSMUSP00000098758.2 Protein coding CCDS20322 Q8BYW1 TSL:1 GENCODE basic APPRIS ALT2

Arhgap25-204 ENSMUST00000145128.7 3816 No protein - Retained intron - - TSL:1

Arhgap25-205 ENSMUST00000203559.1 736 No protein - Retained intron - - TSL:2

94.72 kb Forward strand 87.46Mb 87.48Mb 87.50Mb 87.52Mb 87.54Mb Genes Gm44198-201 >TEC (Comprehensive set...

Contigs AC155726.4 > Genes (Comprehensive set... < Arhgap25-203protein coding

< Arhgap25-204retained intron

< Arhgap25-201protein coding

< Arhgap25-202protein coding

< Arhgap25-205retained intron

Regulatory Build

87.46Mb 87.48Mb 87.50Mb 87.52Mb 87.54Mb Reverse strand 94.72 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000113637

< Arhgap25-203protein coding

Reverse strand 74.69 kb

ENSMUSP00000109... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 Rho GTPase activation protein

SMART Pleckstrin homology domain

Rho GTPase-activating protein domain Pfam Pleckstrin homology domain

Rho GTPase-activating protein domain PROSITE profiles Rho GTPase-activating protein domain

Pleckstrin homology domain PANTHER PTHR15228:SF20

PTHR15228 Gene3D Rho GTPase activation protein

PH-like domain superfamily CDD cd04390

cd13263

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

Variant Legend missense variant stop retained variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 648

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

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