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Mouse Armcx1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Armcx1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Armcx1 gene (NCBI Reference Sequence: NM_001166377 ; Ensembl: ENSMUSG00000033460 ) is located on Mouse chromosome X. 6 exons are identified, with the ATG start codon in exon 6 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000113199). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Armcx1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-275J18 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 6 covers 100.0% of the coding region. Start codon is in exon 6, and stop codon is in exon 6. The size of effective cKO region: ~2726 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 4 5 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Armcx1 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(7368bp) | A(28.07% 2068) | C(18.12% 1335) | T(28.61% 2108) | G(25.2% 1857)

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% chrX + 134717152 134720151 3000 browser details YourSeq 27 424 454 3000 93.6% chr2 - 32664271 32664301 31 browser details YourSeq 25 304 328 3000 100.0% chr19 - 59635442 59635466 25 browser details YourSeq 23 1424 1446 3000 100.0% chr7 - 131841240 131841262 23 browser details YourSeq 22 1424 1445 3000 100.0% chr7 - 56108241 56108262 22 browser details YourSeq 22 1420 1441 3000 100.0% chr3 - 130819810 130819831 22 browser details YourSeq 22 1419 1442 3000 87.0% chr14 - 21964730 21964752 23 browser details YourSeq 20 1802 1827 3000 88.5% chrX - 33345936 33345961 26

Note: The 3000 bp section upstream of Exon 6 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% chrX + 134721520 134724519 3000 browser details YourSeq 1003 1285 2684 3000 96.1% chrX + 134762769 134763830 1062 browser details YourSeq 285 544 2127 3000 91.1% chr17 - 62804743 63178713 373971 browser details YourSeq 255 1698 2128 3000 91.8% chr1 - 179616604 179839398 222795 browser details YourSeq 246 1698 2126 3000 93.1% chr7 + 127342125 127714716 372592 browser details YourSeq 242 1702 2127 3000 92.0% chr11 - 103918138 104537389 619252 browser details YourSeq 235 1698 2126 3000 91.5% chr1 - 20000683 20172485 171803 browser details YourSeq 233 1702 2122 3000 86.2% chr5 + 25524630 25524933 304 browser details YourSeq 225 1708 2126 3000 93.2% chr10 - 128052858 128078597 25740 browser details YourSeq 146 1682 1845 3000 94.6% chr7 - 143155753 143155916 164 browser details YourSeq 138 1683 1845 3000 91.8% chr1 - 87341131 87341291 161 browser details YourSeq 138 1685 1847 3000 95.0% chr12 + 26289898 26290093 196 browser details YourSeq 137 1698 1845 3000 96.7% chr1 + 155589941 155590089 149 browser details YourSeq 136 1685 2052 3000 90.5% chr12 - 12584791 12585223 433 browser details YourSeq 135 1685 1845 3000 92.3% chr11 + 115593322 115593480 159 browser details YourSeq 134 1698 1845 3000 96.0% chr1 - 151328374 151328540 167 browser details YourSeq 134 1698 1849 3000 94.8% chr18 + 32811091 32811263 173 browser details YourSeq 134 1686 1846 3000 89.3% chr11 + 116583397 116583554 158 browser details YourSeq 132 1698 1845 3000 93.2% chr1 + 56709946 56710091 146 browser details YourSeq 131 1698 1845 3000 94.6% chr19 - 41312571 41312720 150

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Armcx1 armadillo repeat containing, X-linked 1 [ Mus musculus (house mouse) ] Gene ID: 78248, updated on 12-Aug-2019

Gene summary

Official Symbol Armcx1 provided by MGI Official Full Name armadillo repeat containing, X-linked 1 provided by MGI Primary source MGI:MGI:1925498 See related Ensembl:ENSMUSG00000033460 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 ALEX1; 3010033I09Rik Expression Broad expression in CNS E18 (RPKM 17.7), CNS E14 (RPKM 16.8) and 16 other tissues See more Orthologs human all

Genomic context

Location: X; X E3 See Armcx1 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (134717913..134721917)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (131252477..131256456)

Chromosome X - NC_000086.7

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Transcript information: This gene has 10 transcripts

Gene: Armcx1 ENSMUSG00000033460

Description armadillo repeat containing, X-linked 1 [Source:MGI Symbol;Acc:MGI:1925498] Gene Synonyms 3010033I09Rik Location Chromosome X: 134,717,963-134,721,917 forward strand. GRCm38:CM001013.2 About this gene This gene has 10 transcripts (splice variants), 142 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Armcx1-205 ENSMUST00000113199.7 2413 456aa ENSMUSP00000108824.1 Protein coding CCDS30398 Q9CX83 TSL:1 GENCODE basic APPRIS P1

Armcx1-201 ENSMUST00000035748.13 2303 456aa ENSMUSP00000043965.7 Protein coding CCDS30398 Q9CX83 TSL:1 GENCODE basic APPRIS P1

Armcx1-202 ENSMUST00000051256.9 2253 456aa ENSMUSP00000053909.3 Protein coding CCDS30398 Q9CX83 TSL:1 GENCODE basic APPRIS P1

Armcx1-206 ENSMUST00000113201.7 2228 456aa ENSMUSP00000108826.1 Protein coding CCDS30398 Q9CX83 TSL:2 GENCODE basic APPRIS P1

Armcx1-203 ENSMUST00000113197.1 2179 456aa ENSMUSP00000108822.1 Protein coding CCDS30398 Q9CX83 TSL:1 GENCODE basic APPRIS P1

Armcx1-204 ENSMUST00000113198.7 2085 456aa ENSMUSP00000108823.1 Protein coding CCDS30398 Q9CX83 TSL:2 GENCODE basic APPRIS P1

Armcx1-209 ENSMUST00000137173.1 951 No protein - lncRNA - - TSL:2

Armcx1-208 ENSMUST00000129697.1 906 No protein - lncRNA - - TSL:5

Armcx1-210 ENSMUST00000138421.7 415 No protein - lncRNA - - TSL:2

Armcx1-207 ENSMUST00000125684.7 320 No protein - lncRNA - - TSL:3

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23.95 kb Forward strand 134.71Mb 134.72Mb 134.73Mb Genes (Comprehensive set... Armcx1-206 >protein coding Gm16397-201 >processed pseudogene

Armcx1-202 >protein coding

Armcx1-208 >lncRNA

Armcx1-205 >protein coding

Armcx1-201 >protein coding

Armcx1-210 >lncRNA

Armcx1-207 >lncRNA

Armcx1-204 >protein coding

Armcx1-203 >protein coding

Armcx1-209 >lncRNA

Contigs BX004852.5 > AL772348.2 > Regulatory Build

134.71Mb 134.72Mb 134.73Mb Reverse strand 23.95 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

pseudogene RNA gene

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Transcript: ENSMUST00000113199

3.92 kb Forward strand

Armcx1-205 >protein coding

ENSMUSP00000108... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Armadillo-type fold SMART Armadillo Pfam Armadillo repeat-containing domain PROSITE profiles PS51257 Armadillo

PANTHER PTHR15712:SF14

PTHR15712 Gene3D Armadillo-like helical

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 456

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

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