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Mouse Kcna6 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Kcna6 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kcna6 gene (NCBI Reference Sequence: NM_013568 ; Ensembl: ENSMUSG00000038077 ) is located on Mouse chromosome 6. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000185333). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kcna6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-131B5 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 mutation of this gene results in an increased thermal nociceptive threshold and in females an increase in circulating triglyceride levels.

Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~1620 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region gRNA region

Wildtype allele A T G T G 5' A 3'

1 2

Targeting vector A T G T G A

Targeted allele A T G T G A

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Kcna6 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(7587bp) | A(23.33% 1770) | C(24.91% 1890) | T(25.58% 1941) | G(26.18% 1986)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr6 - 126739925 126742924 3000 browser details YourSeq 62 2287 2358 3000 97.0% chr5 + 27765617 27766081 465 browser details YourSeq 59 2295 2358 3000 98.4% chr12 - 35308950 35309031 82 browser details YourSeq 59 2295 2358 3000 98.4% chr12 - 35309001 35309157 157 browser details YourSeq 58 2295 2358 3000 98.4% chrX - 101295968 101296076 109 browser details YourSeq 58 2295 2357 3000 98.4% chr12 - 56556642 56556731 90 browser details YourSeq 58 2296 2358 3000 98.4% chr6 + 99075945 99076013 69 browser details YourSeq 57 2295 2357 3000 98.4% chr17 - 81414321 81414476 156 browser details YourSeq 57 2295 2358 3000 96.8% chr1 - 119178504 119178645 142 browser details YourSeq 57 2295 2358 3000 96.8% chr1 - 119178714 119178852 139 browser details YourSeq 57 2295 2358 3000 95.3% chr19 + 53196579 53196648 70 browser details YourSeq 56 2295 2358 3000 95.2% chr10 - 62152129 62152204 76 browser details YourSeq 56 2295 2357 3000 96.7% chr3 + 36296384 36296449 66 browser details YourSeq 55 2295 2357 3000 88.4% chr7 - 130401575 130401634 60 browser details YourSeq 55 2295 2357 3000 88.4% chr1 + 119990327 119990386 60 browser details YourSeq 53 2295 2358 3000 93.6% chr13 + 53558489 53558570 82 browser details YourSeq 53 2295 2358 3000 95.0% chr11 + 3602189 3602272 84 browser details YourSeq 52 2295 2357 3000 92.1% chr13 + 25681703 25681780 78 browser details YourSeq 51 2295 2348 3000 98.2% chr9 - 13621445 13621528 84 browser details YourSeq 51 2295 2358 3000 93.3% chr8 - 86587068 86587166 99

Note: The 3000 bp section upstream of Exon 1 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% chr6 - 126735338 126738337 3000 browser details YourSeq 24 1537 1569 3000 77.0% chr1 + 3163444 3163471 28 browser details YourSeq 22 1258 1279 3000 100.0% chr2 + 165108751 165108772 22 browser details YourSeq 22 1301 1322 3000 100.0% chr1 + 119415420 119415441 22 browser details YourSeq 22 2218 2239 3000 100.0% chr1 + 95085491 95085512 22

Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Kcna6 potassium voltage-gated channel, shaker-related, subfamily, member 6 [ Mus musculus (house mouse) ] Gene ID: 16494, updated on 12-Aug-2019

Gene summary

Official Symbol Kcna6 provided by MGI Official Full Name potassium voltage-gated channel, shaker-related, subfamily, member 6 provided by MGI Primary source MGI:MGI:96663 See related Ensembl:ENSMUSG00000038077 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 Kv1.6; MK1.6 Expression Biased expression in cerebellum adult (RPKM 9.9), cortex adult (RPKM 8.5) and 7 other tissues See more Orthologs human all

Genomic context

Location: 6 F3; 6 61.72 cM See Kcna6 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (126708328..126740834, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (126658347..126690692, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 4 transcripts

Gene: Kcna6 ENSMUSG00000038077

Description potassium voltage-gated channel, shaker-related, subfamily, member 6 [Source:MGI Symbol;Acc:MGI:96663] Gene Synonyms Kv1.6, MK1.6 Location Chromosome 6: 126,708,329-126,740,674 reverse strand. GRCm38:CM000999.2 About this gene This gene has 4 transcripts (splice variants), 81 orthologues, 31 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kcna6- ENSMUST00000185333.1 5836 529aa ENSMUSP00000139481.1 Protein coding CCDS20556 Q61923 TSL:1 204 GENCODE basic APPRIS P1

Kcna6- ENSMUST00000112242.1 3464 529aa ENSMUSP00000107861.1 Protein coding CCDS20556 Q61923 TSL:NA 202 GENCODE basic APPRIS P1

Kcna6- ENSMUST00000040751.5 5827 529aa ENSMUSP00000036872.5 Nonsense mediated - Q61923 TSL:1 201 decay

Kcna6- ENSMUST00000128137.1 2080 No - lncRNA - - TSL:1 203 protein

52.35 kb Forward strand 126.70Mb 126.71Mb 126.72Mb 126.73Mb 126.74Mb 126.75Mb Genes Gm26673-201 >lncRNA (Comprehensive set...

Contigs AC079438.30 > AC124756.4 > Genes (Comprehensive set... < Kcna6-201nonsense mediated decay

< Kcna6-203lncRNA

< Kcna6-204protein coding

< Kcna6-202protein coding

Regulatory Build

126.70Mb 126.71Mb 126.72Mb 126.73Mb 126.74Mb 126.75Mb Reverse strand 52.35 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000185333

< Kcna6-204protein coding

Reverse strand 32.34 kb

ENSMUSP00000139... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily SSF81324

SKP1/BTB/POZ domain superfamily SMART BTB/POZ domain Prints Potassium channel, voltage dependent, Kv

Potassium channel, voltage dependent, Kv1.6

PR00169

Potassium channel, voltage dependent, Kv1 Pfam Potassium channel tetramerisation-type BTB domain

Ion transport domain PANTHER Voltage-gated potassium channel

Potassium channel, voltage dependent, Kv1.6 Gene3D 3.30.710.10 1.10.287.70

CDD cd18407

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 529

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

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