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Mouse Jph2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Jph2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Jph2 (NCBI Reference Sequence: NM_021566 ; Ensembl: ENSMUSG00000017817 ) is located on Mouse 2. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000017961). 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 Jph2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-296M6 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: Homozygotes for a targeted null mutation exhibit a deficiency of junctional membrane complexes in cardiac myocytes and die by embryonic day 10.5.

Exon 3 starts from about 55.17% 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: 34023 bp, and the size of intron 3 for 3'-loxP site insertion: 1489 bp. The size of effective cKO region: ~619 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 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Jph2 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(7119bp) | A(22.49% 1601) | C(25.5% 1815) | T(25.59% 1822) | G(26.42% 1881)

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% chr2 - 163341831 163344830 3000 browser details YourSeq 241 85 556 3000 81.0% chrX + 59626456 59626932 477 browser details YourSeq 207 241 1964 3000 89.5% chr3 - 20159046 20306051 147006 browser details YourSeq 196 115 554 3000 85.0% chr2 + 68516540 68516997 458 browser details YourSeq 193 94 516 3000 87.9% chr7 + 135743722 135744172 451 browser details YourSeq 188 85 474 3000 83.5% chr1 - 127058231 127058611 381 browser details YourSeq 175 120 527 3000 84.6% chr11 - 93578111 93578534 424 browser details YourSeq 175 1804 2111 3000 88.9% chr12 + 31180186 31180522 337 browser details YourSeq 168 207 518 3000 84.5% chr2 - 161821789 161822087 299 browser details YourSeq 167 115 703 3000 83.5% chr1 + 155963535 155964030 496 browser details YourSeq 166 90 480 3000 86.2% chr3 + 36908363 36908775 413 browser details YourSeq 160 111 519 3000 81.0% chr17 + 79053651 79054052 402 browser details YourSeq 159 27 461 3000 83.5% chr11 - 77038396 77038917 522 browser details YourSeq 157 1769 1960 3000 91.7% chr11 + 107557468 107557672 205 browser details YourSeq 155 1780 2105 3000 92.9% chr8 + 35784675 35785365 691 browser details YourSeq 153 697 1071 3000 91.0% chr4 - 129175154 129175675 522 browser details YourSeq 153 1757 1968 3000 91.2% chr15 - 96537938 96538148 211 browser details YourSeq 151 112 461 3000 79.7% chr14 + 73036011 73036362 352 browser details YourSeq 147 116 554 3000 77.6% chr6 + 71758486 71758918 433 browser details YourSeq 146 194 599 3000 78.5% chr17 + 75741784 75742173 390

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% chr2 - 163338212 163341211 3000 browser details YourSeq 36 870 912 3000 92.9% chr1 - 4816482 4816525 44 browser details YourSeq 34 877 911 3000 100.0% chr5 - 115203199 115203234 36 browser details YourSeq 34 871 906 3000 100.0% chr1_GL456221_random - 111084 111132 49 browser details YourSeq 31 877 907 3000 100.0% chr5 - 119277772 119277802 31 browser details YourSeq 29 1698 1726 3000 100.0% chrX - 7375634 7375662 29 browser details YourSeq 29 881 913 3000 83.4% chr6 - 97946471 97946500 30 browser details YourSeq 29 877 906 3000 100.0% chr6 + 145351815 145351846 32 browser details YourSeq 27 881 909 3000 89.3% chr17 - 13223032 13223059 28 browser details YourSeq 27 881 909 3000 89.3% chr17 - 13523629 13523656 28 browser details YourSeq 27 1674 1726 3000 64.6% chr8 + 120229216 120229247 32 browser details YourSeq 27 881 908 3000 100.0% chr1 + 70073190 70073218 29 browser details YourSeq 26 1954 1983 3000 93.4% chr5 + 73399262 73399291 30 browser details YourSeq 25 1918 1945 3000 96.5% chr5 - 99667841 99667869 29 browser details YourSeq 24 2344 2367 3000 100.0% chr3 - 145883768 145883791 24 browser details YourSeq 24 2064 2087 3000 100.0% chr8 + 23682365 23682388 24 browser details YourSeq 23 2868 2893 3000 96.0% chr17 - 47258401 47258426 26 browser details YourSeq 23 878 900 3000 100.0% chr15 - 67624430 67624452 23 browser details YourSeq 23 872 895 3000 100.0% chr1 + 15664468 15664496 29 browser details YourSeq 22 2178 2199 3000 100.0% chr18 - 46166950 46166971 22

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 information: Jph2 junctophilin 2 [ Mus musculus (house mouse) ] Gene ID: 59091, updated on 10-Oct-2019

Gene summary

Official Symbol Jph2 provided by MGI Official Full Name junctophilin 2 provided by MGI Primary source MGI:MGI:1891496 See related Ensembl:ENSMUSG00000017817 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 Jp2; JP-2; 1110002E14Rik Expression Biased expression in heart adult (RPKM 70.1), bladder adult (RPKM 48.2) and 10 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 H2-H3 See Jph2 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (163336242..163398049, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (163163509..163223686, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 2 transcripts

Gene: Jph2 ENSMUSG00000017817

Description junctophilin 2 [Source:MGI Symbol;Acc:MGI:1891496] Gene Synonyms 1110002E14Rik, JP-2 Location Chromosome 2: 163,336,242-163,397,993 reverse strand. GRCm38:CM000995.2 About this gene This gene has 2 transcripts (splice variants), 198 orthologues, 3 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

Jph2-201 ENSMUST00000017961.10 4211 696aa ENSMUSP00000017961.4 Protein coding CCDS17008 Q9ET78 TSL:1 GENCODE basic APPRIS P1

Jph2-202 ENSMUST00000109425.2 4155 696aa ENSMUSP00000105052.2 Protein coding CCDS17008 Q9ET78 TSL:1 GENCODE basic APPRIS P1

81.75 kb Forward strand 163.34Mb 163.36Mb 163.38Mb 163.40Mb Contigs AL589876.11 > (Comprehensive set... < Jph2-202protein coding < Oser1-206lncRNA

< Jph2-201protein coding < Oser1-205lncRNA

< Oser1-201protein coding

< Oser1-202protein coding

< Oser1-204protein coding

< Oser1-207lncRNA

Regulatory Build

163.34Mb 163.36Mb 163.38Mb 163.40Mb Reverse strand 81.75 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000017961

< Jph2-201protein coding

Reverse strand 61.71 kb

ENSMUSP00000017... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily SSF82185 SMART MORN motif Pfam MORN motif PIRSF Junctophilin

PANTHER Junctophilin

PTHR23085:SF26 Gene3D 2.20.110.10

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 696

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

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