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Mouse Kcnip2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Kcnip2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kcnip2 gene (NCBI Reference Sequence: NM_145703 ; Ensembl: ENSMUSG00000025221 ) is located on Mouse chromosome 19. 10 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000162528). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kcnip2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-98F2 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 disruptions in this gene are susceptible to induced cardiac arrhythmias but are otherwise normal.

Exon 4 starts from about 27.65% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 574 bp, and the size of intron 4 for 3'-loxP site insertion: 532 bp. The size of effective cKO region: ~625 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 2 3 4 5 6 7 8 9 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Kcnip2 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(7125bp) | A(21.77% 1551) | C(25.31% 1803) | T(25.74% 1834) | G(27.19% 1937)

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% chr19 - 45795955 45798954 3000 browser details YourSeq 112 903 1033 3000 94.6% chr10 - 61454222 61454701 480 browser details YourSeq 106 903 1039 3000 90.9% chr5 + 135669679 135669834 156 browser details YourSeq 104 900 1039 3000 92.8% chr12 - 4142109 4541191 399083 browser details YourSeq 103 903 1028 3000 93.4% chr13 - 64330716 64339256 8541 browser details YourSeq 103 906 1039 3000 93.4% chr10 + 39146710 39146875 166 browser details YourSeq 102 905 1033 3000 91.9% chr2 - 155986655 155986790 136 browser details YourSeq 102 902 1040 3000 90.4% chr1 + 178272085 178272670 586 browser details YourSeq 101 905 1028 3000 91.8% chr16 + 4490692 4490826 135 browser details YourSeq 100 900 1039 3000 93.2% chr19 - 29665418 29665563 146 browser details YourSeq 100 906 1029 3000 94.0% chr14 - 124437092 124437232 141 browser details YourSeq 100 903 1035 3000 93.9% chr14 - 67227359 67227493 135 browser details YourSeq 100 919 1035 3000 93.2% chr10 - 79700579 79700701 123 browser details YourSeq 100 904 1035 3000 91.8% chr9 + 40262399 40262540 142 browser details YourSeq 99 903 1035 3000 89.6% chr4 - 127106252 127106388 137 browser details YourSeq 99 904 1026 3000 91.0% chrX + 52222054 52222189 136 browser details YourSeq 99 918 1036 3000 94.0% chr12 + 86406079 86406209 131 browser details YourSeq 99 919 1038 3000 96.3% chr11 + 59246837 59246966 130 browser details YourSeq 98 915 1028 3000 93.9% chr17 - 29390786 29390912 127 browser details YourSeq 98 918 1039 3000 91.0% chr4 + 5890631 5890770 140

Note: The 3000 bp section upstream of Exon 4 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% chr19 - 45792330 45795329 3000 browser details YourSeq 32 1536 1582 3000 97.2% chr13 - 37653683 37653730 48 browser details YourSeq 29 645 676 3000 96.8% chr5 + 131892381 131892412 32 browser details YourSeq 28 1215 1246 3000 96.7% chr10 - 71043656 71043706 51 browser details YourSeq 28 641 675 3000 75.9% chr13 + 35161126 35161154 29 browser details YourSeq 27 1211 1242 3000 93.6% chr2 - 21777076 21777116 41 browser details YourSeq 26 652 677 3000 100.0% chr5 + 36813226 36813251 26 browser details YourSeq 25 650 676 3000 88.5% chr13 + 51728166 51728191 26 browser details YourSeq 25 644 670 3000 100.0% chr10 + 62547832 62547861 30 browser details YourSeq 22 641 662 3000 100.0% chr1 - 100365820 100365841 22 browser details YourSeq 22 2271 2292 3000 100.0% chr8 + 123151126 123151147 22 browser details YourSeq 21 404 424 3000 100.0% chr2 + 69875361 69875381 21

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Kcnip2 Kv channel-interacting protein 2 [ Mus musculus (house mouse) ] Gene ID: 80906, updated on 7-Oct-2019

Gene summary

Official Symbol Kcnip2 provided by MGI Official Full Name Kv channel-interacting protein 2 provided by MGI Primary source MGI:MGI:2135916 See related Ensembl:ENSMUSG00000025221 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as KChIP2 Summary This gene encodes a member of the voltage-gated potassium channel-interacting protein (KCNIP) family. KCNIP family Expression members are small calcium binding proteins that commonly exhibit unique variation at their N-termini, and which modulate A-type potassium channels. This gene is predominantly expressed in the adult heart, and to a lesser extent in the brain. Disruption of this gene is associated with susceptibility to cardiac arrhythmias and lack of transient outward potassium current in ventricular myocytes, and downregulated expression is associated with cardiac hypertrophy. The encoded protein has also been implicated as a repressor of immune response. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Feb 2013] Orthologs Biased expression in heart adult (RPKM 60.6), cortex adult (RPKM 30.5) and 5 other tissuesS ee more human all

Genomic context

Location: 19 C3; 19 38.75 cM See Kcnip2 in Genome Data Viewer Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (45792346..45816422, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (45868160..45890293, complement)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 11 transcripts

Gene: Kcnip2 ENSMUSG00000025221

Description Kv channel-interacting protein 2 [Source:MGI Symbol;Acc:MGI:2135916] Gene Synonyms KChIP2 Location Chromosome 19: 45,791,839-45,816,061 reverse strand. GRCm38:CM001012.2 About this gene This gene has 11 transcripts (splice variants), 245 orthologues, 14 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kcnip2- ENSMUST00000162528.8 2395 270aa ENSMUSP00000125142.2 Protein CCDS38004 Q3YAB3 TSL:1 209 coding GENCODE basic APPRIS ALT1

Kcnip2- ENSMUST00000079431.9 2302 252aa ENSMUSP00000078400.3 Protein CCDS50453 Q3YAB2 TSL:1 202 coding GENCODE basic APPRIS ALT1

Kcnip2- ENSMUST00000086993.10 1005 225aa ENSMUSP00000084215.4 Protein CCDS70955 Q3YAA4 TSL:1 203 coding Q9JJ69 GENCODE basic

Kcnip2- ENSMUST00000161886.8 693 220aa ENSMUSP00000124482.2 Protein CCDS29868 Q3YAB1 TSL:1 208 coding GENCODE basic APPRIS P3

Kcnip2- ENSMUST00000239049.1 2113 230aa ENSMUSP00000158890.1 Protein - - APPRIS ALT1 211 coding

Kcnip2- ENSMUST00000026247.12 1203 252aa ENSMUSP00000026247.6 Protein - E9QNK8 TSL:5 201 coding GENCODE basic APPRIS ALT1

Kcnip2- ENSMUST00000159245.7 519 172aa ENSMUSP00000124346.1 Protein - F6TBA6 CDS 5' incomplete 206 coding TSL:5

Kcnip2- ENSMUST00000162661.8 517 172aa ENSMUSP00000124821.3 Protein - E0CX94 CDS 3' incomplete 210 coding TSL:5

Kcnip2- ENSMUST00000159210.8 383 95aa ENSMUSP00000124763.2 Protein - E0CXC0 CDS 5' incomplete 205 coding TSL:3

Kcnip2- ENSMUST00000159446.1 375 125aa ENSMUSP00000125499.1 Protein - F7BWB2 CDS 5' and 3' 207 coding incomplete TSL:3

Kcnip2- ENSMUST00000111906.1 3114 No - Retained - - TSL:2 204 protein intron

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44.22 kb Forward strand 45.79Mb 45.80Mb 45.81Mb 45.82Mb Contigs AC149086.4 > AC131185.4 > Genes (Comprehensive set... < Oga-201protein coding < Kcnip2-205protein coding < Armh3-201protein coding

< Oga-205retained intron < Kcnip2-202protein coding

< Oga-202protein coding < Kcnip2-209protein coding

< Kcnip2-204retained intron

< Kcnip2-211protein coding

< Kcnip2-203protein coding

< Kcnip2-201protein coding

< Kcnip2-208protein coding

< Kcnip2-206protein coding

< Kcnip2-210protein coding

< Kcnip2-207protein coding

Regulatory Build

45.79Mb 45.80Mb 45.81Mb 45.82Mb Reverse strand 44.22 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000162528

< Kcnip2-209protein coding

Reverse strand 23.72 kb

ENSMUSP00000125... MobiDB lite Low complexity (Seg) Superfamily EF-hand domain pair

SMART EF-hand domain Prints PR00450 Pfam EF-hand domain EF-hand domain

PROSITE profiles EF-hand domain PROSITE patterns EF-Hand 1, calcium-binding site PANTHER PTHR23055:SF65

Recoverin family Gene3D 1.10.238.10 CDD EF-hand domain

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

Variant Legend

synonymous variant

Scale bar 0 40 80 120 160 200 270

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

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