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Mouse Kcnj9 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Kcnj9 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kcnj9 gene (NCBI Reference Sequence: NM_008429 ; Ensembl: ENSMUSG00000038026 ) is located on Mouse chromosome 1. 4 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000062387). 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 Kcnj9 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-82L21 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: Inactivation of this locus does not result in any overt phenotype.

Exon 3 starts from about 100% 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: 1414 bp, and the size of intron 3 for 3'-loxP site insertion: 2511 bp. The size of effective cKO region: ~1350 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Kcnj9 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. 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(7850bp) | A(23.54% 1848) | C(24.94% 1958) | T(23.25% 1825) | G(28.27% 2219)

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% chr1 - 172326806 172329805 3000 browser details YourSeq 152 2675 2872 3000 90.5% chr5 - 3476790 3476993 204 browser details YourSeq 152 2654 2872 3000 83.4% chr10 + 61573331 61573528 198 browser details YourSeq 150 2666 2871 3000 87.7% chr4 - 59409269 59409473 205 browser details YourSeq 148 2658 2872 3000 82.8% chr11 - 108888556 108888753 198 browser details YourSeq 145 2687 2877 3000 90.2% chr6 - 120028456 120028650 195 browser details YourSeq 145 2680 2883 3000 84.2% chr6 - 87037397 87037597 201 browser details YourSeq 145 2682 2872 3000 90.5% chr4 - 57919174 57919372 199 browser details YourSeq 145 2660 2872 3000 83.3% chr17 - 6588167 6588354 188 browser details YourSeq 145 2684 2872 3000 88.8% chr14 + 34282789 34282977 189 browser details YourSeq 144 2689 2872 3000 88.4% chr1 - 105769142 105769323 182 browser details YourSeq 142 2686 2872 3000 89.1% chr10 + 118124771 118124957 187 browser details YourSeq 141 2677 2872 3000 86.1% chr8 + 43256621 43256809 189 browser details YourSeq 141 2683 2872 3000 88.3% chr5 + 108067129 108067337 209 browser details YourSeq 140 2695 2889 3000 85.8% chr17 - 74503769 74503952 184 browser details YourSeq 139 2688 2869 3000 87.2% chr4 - 149209611 149209790 180 browser details YourSeq 139 2689 2872 3000 89.8% chr17 - 36028357 36028548 192 browser details YourSeq 139 2688 2872 3000 89.3% chr5 + 104119132 104119317 186 browser details YourSeq 139 2687 2873 3000 88.0% chr4 + 116873955 116874141 187 browser details YourSeq 138 2688 2870 3000 85.3% chr9 - 106131920 106132095 176

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% chr1 - 172322456 172325455 3000 browser details YourSeq 46 2240 2326 3000 72.4% chr16 - 94762621 94762700 80 browser details YourSeq 46 1788 1882 3000 81.3% chr13 - 55928844 55928936 93 browser details YourSeq 46 1809 1922 3000 96.0% chr14 + 70280791 70280931 141 browser details YourSeq 45 1788 1890 3000 89.5% chr6 + 93584801 93584904 104 browser details YourSeq 44 1776 1886 3000 78.0% chr12 + 117208847 117208946 100 browser details YourSeq 41 1771 1890 3000 93.7% chr1 - 85811136 85811273 138 browser details YourSeq 41 1771 1887 3000 91.2% chr6 + 141092102 141092217 116 browser details YourSeq 40 1861 2141 3000 95.5% chr6 - 94805117 94805438 322 browser details YourSeq 40 1860 1925 3000 87.0% chr13 + 52308594 52308657 64 browser details YourSeq 36 1788 1875 3000 86.4% chr1 - 91660651 91660737 87 browser details YourSeq 36 1788 1889 3000 89.2% chr12 + 17761001 17761103 103 browser details YourSeq 35 1790 1874 3000 87.2% chr10 - 26711955 26712037 83 browser details YourSeq 34 1788 1885 3000 92.5% chr16 - 35282082 35282181 100 browser details YourSeq 34 1861 1900 3000 97.3% chr11 - 68086499 68086540 42 browser details YourSeq 34 1858 1891 3000 100.0% chr7 + 121781267 121781300 34 browser details YourSeq 33 1854 1887 3000 100.0% chr6 + 23106204 23106239 36 browser details YourSeq 33 1856 1889 3000 100.0% chr2 + 166983131 166983165 35 browser details YourSeq 32 1810 1880 3000 88.3% chr16 + 5104305 5104373 69 browser details YourSeq 30 1788 1884 3000 88.3% chrX - 152925761 152925856 96

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: Kcnj9 potassium inwardly-rectifying channel, subfamily J, member 9 [ Mus musculus (house mouse) ] Gene ID: 16524, updated on 24-Oct-2019

Gene summary

Official Symbol Kcnj9 provided by MGI Official Full Name potassium inwardly-rectifying channel, subfamily J, member 9 provided by MGI Primary source MGI:MGI:108007 See related Ensembl:ENSMUSG00000038026 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 Girk3; Kir3.3; mbGIRK3; 1700085N21Rik Expression Biased expression in cortex adult (RPKM 37.4), cerebellum adult (RPKM 26.5) and 3 other tissuesS ee more Orthologs human all

Genomic context

Location: 1 H3; 1 79.66 cM See Kcnj9 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (172320508..172329322, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (174251164..174259394, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 3 transcripts

Gene: Kcnj9 ENSMUSG00000038026

Description potassium inwardly-rectifying channel, subfamily J, member 9 [Source:MGI Symbol;Acc:MGI:108007] Gene Synonyms 1700085N21Rik, Girk3, Kir3.3 Location Chromosome 1: 172,320,501-172,329,318 reverse strand. GRCm38:CM000994.2 About this gene This gene has 3 transcripts (splice variants), 175 orthologues, 14 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kcnj9-201 ENSMUST00000062387.7 3857 393aa ENSMUSP00000060110.2 Protein coding CCDS35783 P48543 Q544N3 TSL:1 GENCODE basic APPRIS P1

Kcnj9-202 ENSMUST00000194204.1 2265 393aa ENSMUSP00000141633.1 Protein coding CCDS35783 P48543 Q544N3 TSL:1 GENCODE basic APPRIS P1

Kcnj9-203 ENSMUST00000194860.1 1390 No protein - Retained intron - - TSL:5

28.82 kb Forward strand 172.315Mb 172.320Mb 172.325Mb 172.330Mb 172.335Mb Genes Igsf8-204 >protein coding Gm36937-201 >lncRNA (Comprehensive set...

Igsf8-202 >protein coding

Igsf8-203 >protein coding Igsf8-207 >protein coding

Igsf8-201 >protein coding

Igsf8-208 >protein coding

Igsf8-205 >lncRNA

Contigs < AC087061.22 AC074311.28 > Genes (Comprehensive set... < Kcnj9-201protein coding

< Kcnj9-202protein coding

< Kcnj9-203retained intron

Regulatory Build

172.315Mb 172.320Mb 172.325Mb 172.330Mb 172.335Mb Reverse strand 28.82 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000062387

< Kcnj9-201protein coding

Reverse strand 8.82 kb

ENSMUSP00000060... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily SSF81324

Immunoglobulin E-set Prints Potassium channel, inwardly rectifying, Kir3.3

Potassium channel, inwardly rectifying, Kir Pfam Potassium channel, inwardly rectifying, transmembrane domain

Inward rectifier potassium channel, C-terminal PIRSF Potassium channel, inwardly rectifying, Kir PANTHER Potassium channel, inwardly rectifying, Kir3.3

Potassium channel, inwardly rectifying, Kir Gene3D Potassium channel, inwardly rectifying, Kir, cytoplasmic

1.10.287.70

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 393

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

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