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Mouse Kcnu1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Kcnu1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kcnu1 gene (NCBI Reference Sequence: NM_008432 ; Ensembl: ENSMUSG00000031576 ) is located on Mouse chromosome 8. 27 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 27 (Transcript: ENSMUST00000098858). 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 Kcnu1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-269L13 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: Homozygous male mutants are infertile with impaired sperm capacitation.

Exon 3 starts from about 9.4% 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: 6179 bp, and the size of intron 3 for 3'-loxP site insertion: 1038 bp. The size of effective cKO region: ~562 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 3 4 27 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Kcnu1 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(7062bp) | A(28.77% 2032) | C(22.3% 1575) | T(27.43% 1937) | G(21.5% 1518)

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% chr8 + 25854874 25857873 3000 browser details YourSeq 397 1 711 3000 92.0% chr6 + 134496699 134497561 863 browser details YourSeq 396 1 715 3000 94.7% chr3 - 68831872 68832725 854 browser details YourSeq 388 1 702 3000 93.3% chr5 + 92640583 92641441 859 browser details YourSeq 338 1 375 3000 96.0% chr12 - 3945740 3946305 566 browser details YourSeq 337 1 380 3000 94.0% chr2 + 19167408 19167786 379 browser details YourSeq 334 1 380 3000 94.9% chr3 + 50843206 50843585 380 browser details YourSeq 333 1 362 3000 96.2% chr7 - 87044895 87045257 363 browser details YourSeq 333 1 362 3000 96.2% chr7 - 87046387 87046749 363 browser details YourSeq 333 1 364 3000 95.9% chrX + 100722313 100722677 365 browser details YourSeq 333 1 362 3000 96.2% chr2 + 176716170 176716532 363 browser details YourSeq 333 1 362 3000 96.2% chr2 + 175973178 175973540 363 browser details YourSeq 333 1 362 3000 96.2% chr2 + 175648214 175648576 363 browser details YourSeq 332 1 362 3000 97.8% chrX + 144298637 144298999 363 browser details YourSeq 332 1 362 3000 95.9% chr19 + 21966603 21966964 362 browser details YourSeq 331 1 362 3000 95.9% chr2 + 175744280 175744642 363 browser details YourSeq 331 1 362 3000 95.9% chr2 + 175745773 175746135 363 browser details YourSeq 331 1 362 3000 95.9% chr2 + 175971685 175972047 363 browser details YourSeq 331 1 362 3000 95.9% chr2 + 176714677 176715039 363 browser details YourSeq 331 1 362 3000 95.9% chr2 + 175646721 175647083 363

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% chr8 + 25858436 25861435 3000 browser details YourSeq 236 1 292 3000 90.5% chr5 + 146889974 146890265 292 browser details YourSeq 235 1 273 3000 92.3% chr12 - 111399672 111399943 272 browser details YourSeq 234 1 309 3000 88.3% chr6 - 89229741 89230049 309 browser details YourSeq 234 1 288 3000 89.9% chr7 + 24121685 24121971 287 browser details YourSeq 233 1 288 3000 91.0% chr9 + 45214745 45215052 308 browser details YourSeq 233 1 288 3000 89.2% chr14 + 118830011 118830295 285 browser details YourSeq 232 1 286 3000 93.0% chr9 - 122322561 122323192 632 browser details YourSeq 230 1 296 3000 88.1% chr5 - 107998469 107998762 294 browser details YourSeq 230 5 292 3000 89.2% chr8 + 119968782 119969068 287 browser details YourSeq 230 1 288 3000 91.4% chr10 + 70646178 70646484 307 browser details YourSeq 229 1 287 3000 89.2% chr1 - 157108881 157109166 286 browser details YourSeq 228 1 288 3000 88.9% chr2 - 80607569 80607855 287 browser details YourSeq 228 1 286 3000 89.2% chr17 - 88299071 88299355 285 browser details YourSeq 228 1 288 3000 88.9% chr14 + 78920518 78920804 287 browser details YourSeq 228 1 288 3000 88.5% chr1 + 90438457 90438742 286 browser details YourSeq 227 1 276 3000 91.4% chrX - 94092941 94336858 243918 browser details YourSeq 227 1 287 3000 88.9% chr2 + 169201466 169201751 286 browser details YourSeq 227 1 288 3000 89.6% chr11 + 113321096 113321384 289 browser details YourSeq 226 1 296 3000 87.5% chr9 - 48011621 48011915 295

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 protein information: Kcnu1 potassium channel, subfamily U, member 1 [ Mus musculus (house mouse) ] Gene ID: 16532, updated on 10-Oct-2019

Gene summary

Official Symbol Kcnu1 provided by MGI Official Full Name potassium channel, subfamily U, member 1 provided by MGI Primary source MGI:MGI:1202300 See related Ensembl:ENSMUSG00000031576 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 Slo3; mSlo3; Kcnma3 Expression Biased expression in testis adult (RPKM 17.3) and CNS E18 (RPKM 1.2) See more Orthologs human all

Genomic context

Location: 8; 8 A2 See Kcnu1 in Genome Data Viewer

Exon count: 29

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (25848627..25937943)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (26960095..27048406)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 4 transcripts

Gene: Kcnu1 ENSMUSG00000031576

Description potassium channel, subfamily U, member 1 [Source:MGI Symbol;Acc:MGI:1202300] Gene Synonyms Kcnma3, Slo3 Location Chromosome 8: 25,849,623-25,937,939 forward strand. GRCm38:CM001001.2 About this gene This gene has 4 transcripts (splice variants), 107 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kcnu1-201 ENSMUST00000098858.10 3536 1121aa ENSMUSP00000096457.3 Protein coding CCDS52530 G3X9P7 TSL:1 GENCODE basic APPRIS P1

Kcnu1-202 ENSMUST00000120653.7 1446 381aa ENSMUSP00000113442.1 Protein coding - D3Z5P2 TSL:1 GENCODE basic

Kcnu1-203 ENSMUST00000126226.1 697 160aa ENSMUSP00000123273.1 Protein coding - D3Z073 CDS 3' incomplete TSL:3

Kcnu1-204 ENSMUST00000210273.1 549 No protein - Retained intron - - TSL:5

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108.32 kb Forward strand 25.84Mb 25.86Mb 25.88Mb 25.90Mb 25.92Mb 25.94Mb Genes (Comprehensive set... Kcnu1-201 >protein coding

Kcnu1-204 >retained intron Kcnu1-202 >protein coding

Kcnu1-203 >protein coding

Contigs AC122752.10 > Genes < Ash2l-201protein coding < Hgsnat-203retained intron (Comprehensive set...

< Ash2l-205nonsense mediated decay < Hgsnat-201protein coding

< Ash2l-210protein coding

< Ash2l-208nonsense mediated decay

< Ash2l-204protein coding

< Ash2l-203protein coding

< Ash2l-202protein coding

Regulatory Build

25.84Mb 25.86Mb 25.88Mb 25.90Mb 25.92Mb 25.94Mb Reverse strand 108.32 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000098858

88.32 kb Forward strand

Kcnu1-201 >protein coding

ENSMUSP00000096... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily SSF81324 Prints PR00169 PR01449 Pfam Ion transport domain Calcium-activated potassium channel BK, alpha subunit

PANTHER PTHR10027

PTHR10027:SF23 Gene3D Voltage-dependent channel domain superfamily

1.10.287.70 3.40.50.720

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

Variant Legend stop gained inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1121

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

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