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Mouse Arcn1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Arcn1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Arcn1 gene (NCBI Reference Sequence: NM_145985 ; Ensembl: ENSMUSG00000032096 ) is located on Mouse chromosome 9. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000034607). Exon 3~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Arcn1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-173C11 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 spontaneous mutation have a dilute coat color and neurological defects.

Exon 3 starts from about 17.48% of the coding region. The knockout of Exon 3~5 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 901 bp, and the size of intron 5 for 3'-loxP site insertion: 4844 bp. The size of effective cKO region: ~2441 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 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Arcn1 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(8941bp) | A(28.39% 2538) | C(19.21% 1718) | T(32.46% 2902) | G(19.94% 1783)

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% chr9 - 44759311 44762310 3000 browser details YourSeq 257 2085 2349 3000 98.5% chr17 + 35400801 35401065 265 browser details YourSeq 257 2085 2349 3000 98.5% chr17 + 35445996 35446260 265 browser details YourSeq 180 2665 2893 3000 94.2% chrX - 151636294 151636543 250 browser details YourSeq 172 2666 2871 3000 91.3% chr2 - 122622465 122622664 200 browser details YourSeq 171 2682 2870 3000 95.8% chr1 - 191948240 191948436 197 browser details YourSeq 171 2684 2869 3000 96.3% chr4 + 129751410 129751600 191 browser details YourSeq 170 2685 2870 3000 96.3% chr11 - 104124166 104323481 199316 browser details YourSeq 170 2674 2870 3000 93.9% chr11 - 29633538 29633756 219 browser details YourSeq 170 2687 2875 3000 95.3% chr6 + 64735785 64736154 370 browser details YourSeq 169 2682 2871 3000 94.8% chr11 + 61625968 61626159 192 browser details YourSeq 169 2666 2876 3000 89.9% chr10 + 56345936 56346140 205 browser details YourSeq 168 2665 2877 3000 88.3% chr15 + 3379573 3379768 196 browser details YourSeq 167 2685 2869 3000 95.7% chr2 + 58624056 58624243 188 browser details YourSeq 166 2685 2896 3000 90.0% chr2 + 152853830 152854030 201 browser details YourSeq 166 2697 2994 3000 92.4% chr11 + 34011275 34011603 329 browser details YourSeq 165 2682 2877 3000 92.9% chrX - 8104029 8104231 203 browser details YourSeq 165 2685 2869 3000 95.2% chr12 - 111513283 111513470 188 browser details YourSeq 165 2682 2867 3000 94.7% chr11 - 4244595 4244783 189 browser details YourSeq 165 2683 2871 3000 95.1% chr8 + 47597882 47598071 190

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% chr9 - 44753870 44756869 3000 browser details YourSeq 356 1067 2580 3000 92.4% chr4 + 124272826 124682085 409260 browser details YourSeq 353 1119 2653 3000 93.0% chr11 - 95955559 96360923 405365 browser details YourSeq 189 2388 3000 3000 86.7% chr11 + 109307187 109307411 225 browser details YourSeq 180 2409 2656 3000 92.9% chr18 - 56654860 56655093 234 browser details YourSeq 180 2392 2593 3000 96.0% chr7 + 89470178 89470558 381 browser details YourSeq 176 2387 2580 3000 94.3% chr13 - 106922802 106922992 191 browser details YourSeq 174 2387 2597 3000 91.1% chr10 - 49577745 49577952 208 browser details YourSeq 174 2389 2582 3000 94.3% chr1 + 47168305 47168497 193 browser details YourSeq 173 2392 2596 3000 92.2% chr1 + 85808100 85808297 198 browser details YourSeq 172 2391 2582 3000 94.7% chr19 - 24931236 24931425 190 browser details YourSeq 171 2288 2580 3000 87.9% chr1 + 86170662 86170875 214 browser details YourSeq 170 2389 2581 3000 94.0% chrX + 48631242 48631431 190 browser details YourSeq 169 2386 2581 3000 93.6% chrX - 52672681 52672873 193 browser details YourSeq 168 2388 2580 3000 93.7% chr15 - 102652182 102652372 191 browser details YourSeq 167 2390 2581 3000 92.4% chr4 + 147642766 147642951 186 browser details YourSeq 166 2391 2581 3000 92.4% chr4 - 147360866 147361050 185 browser details YourSeq 165 2390 2581 3000 92.9% chr11 - 106210796 106210984 189 browser details YourSeq 164 2401 2580 3000 94.4% chr12 - 31478447 31478623 177 browser details YourSeq 159 2388 2584 3000 89.9% chr16 - 8581124 8581308 185

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Arcn1 archain 1 [ Mus musculus (house mouse) ] Gene ID: 213827, updated on 12-Aug-2019

Gene summary

Official Symbol Arcn1 provided by MGI Official Full Name archain 1 provided by MGI Primary source MGI:MGI:2387591 See related Ensembl:ENSMUSG00000032096 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as nur17; 4632432M07Rik Expression Ubiquitous expression in placenta adult (RPKM 54.6), limb E14.5 (RPKM 40.9) and 28 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 A5.2 See Arcn1 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (44742143..44767808, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (44550226..44575891, complement)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 6 transcripts

Gene: Arcn1 ENSMUSG00000032096

Description archain 1 [Source:MGI Symbol;Acc:MGI:2387591] Gene Synonyms 4632432M07Rik, delta-COP, nur17, pale coat neuro Location Chromosome 9: 44,741,564-44,767,845 reverse strand. GRCm38:CM001002.2 About this gene This gene has 6 transcripts (splice variants), 214 orthologues, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Arcn1-201 ENSMUST00000034607.9 4072 511aa ENSMUSP00000034607.9 Protein coding CCDS40602 Q5XJY5 TSL:1 GENCODE basic APPRIS P1

Arcn1-204 ENSMUST00000215140.1 3497 No protein - Retained intron - - TSL:NA

Arcn1-206 ENSMUST00000217199.1 3211 No protein - Retained intron - - TSL:NA

Arcn1-203 ENSMUST00000150160.7 2956 No protein - Retained intron - - TSL:1

Arcn1-205 ENSMUST00000217194.1 2616 No protein - Retained intron - - TSL:NA

Arcn1-202 ENSMUST00000125164.1 739 No protein - lncRNA - - TSL:5

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46.28 kb Forward strand

44.74Mb 44.75Mb 44.76Mb 44.77Mb Genes Ift46-212 >lncRNA (Comprehensive set...

Ift46-201 >protein coding

Ift46-205 >protein coding

Ift46-215 >protein coding

Ift46-213 >protein coding

Ift46-204 >protein coding

Ift46-214 >protein coding

Ift46-209 >protein coding

Ift46-211 >protein coding

Ift46-202 >protein coding

Ift46-208 >lncRNA

Ift46-203 >lncRNA

Contigs AC142113.2 > Genes (Comprehensive set... < Phldb1-201protein coding < Arcn1-201protein coding

< Gm24166-201snoRNA < Arcn1-205retained intron < Arcn1-203retained intron

< Gm48562-201TEC < Arcn1-206retained intron

< Arcn1-202lncRNA < Arcn1-204retained intron

Regulatory Build

44.74Mb 44.75Mb 44.76Mb 44.77Mb Reverse strand 46.28 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000034607

< Arcn1-201protein coding

Reverse strand 26.28 kb

ENSMUSP00000034... PDB-ENSP mappings Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Longin-like domain superfamily AP-2 complex subunit mu, C-terminal superfamily

Pfam AP complex, mu/sigma subunit Mu homology domain

PROSITE profiles Mu homology domain

PANTHER Coatomer delta subunit

PTHR10121:SF0 Gene3D 3.30.450.60 2.60.40.1170

CDD cd14830 cd09254

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

Variant Legend synonymous variant

Scale bar 0 60 120 180 240 300 360 420 511

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

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