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Mouse Ttyh2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ttyh2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ttyh2 gene (NCBI Reference Sequence: NM_053273 ; Ensembl: ENSMUSG00000034714 ) is located on Mouse chromosome 11. 14 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 14 (Transcript: ENSMUST00000045779). 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 Ttyh2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-84I19 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:

Exon 3 starts from about 18.98% 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: 3672 bp, and the size of intron 3 for 3'-loxP site insertion: 6238 bp. The size of effective cKO region: ~612 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 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ttyh2 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(7112bp) | A(23.73% 1688) | C(24.18% 1720) | T(25.94% 1845) | G(26.14% 1859)

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% chr11 + 114686990 114689989 3000 browser details YourSeq 338 1444 2379 3000 91.5% chr13 + 13362765 13419628 56864 browser details YourSeq 290 1444 2613 3000 91.7% chr4 - 108280145 108306365 26221 browser details YourSeq 279 2102 2751 3000 89.8% chr2 + 32886720 32887429 710 browser details YourSeq 256 2104 2719 3000 85.1% chr1 + 85039974 85040491 518 browser details YourSeq 247 2108 2752 3000 90.6% chr8 + 104016074 104416972 400899 browser details YourSeq 244 2104 2752 3000 91.0% chr3 + 145489269 145489957 689 browser details YourSeq 237 2102 2389 3000 92.0% chr12 - 108369947 108370302 356 browser details YourSeq 234 2104 2735 3000 92.2% chr8 + 72575624 72576288 665 browser details YourSeq 232 2115 2743 3000 86.2% chr15 - 29621671 29622116 446 browser details YourSeq 231 2102 2614 3000 89.1% chr16 + 50506758 50507482 725 browser details YourSeq 225 2102 2393 3000 91.0% chr14 - 101744095 101744443 349 browser details YourSeq 225 2108 2398 3000 90.7% chr12 - 72977549 72977886 338 browser details YourSeq 225 2082 2393 3000 89.8% chr1 - 75459888 75460226 339 browser details YourSeq 224 2108 2403 3000 91.0% chr5 - 107607551 107607884 334 browser details YourSeq 223 2108 2392 3000 90.6% chr5 - 134355712 134356017 306 browser details YourSeq 223 2060 2390 3000 90.3% chr17 - 84069428 84069763 336 browser details YourSeq 221 2082 2388 3000 89.0% chr8 + 3326569 3326873 305 browser details YourSeq 220 2117 2392 3000 91.1% chr9 - 73077867 73078181 315 browser details YourSeq 220 2082 2462 3000 90.2% chr8 + 123818187 123818849 663

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% chr11 + 114690602 114693601 3000 browser details YourSeq 43 2731 2791 3000 96.0% chr2 - 93347903 93347964 62 browser details YourSeq 42 2730 2794 3000 87.5% chr13 + 61242685 61242747 63 browser details YourSeq 39 2738 2791 3000 83.8% chr15 + 11170509 11170557 49 browser details YourSeq 38 2702 2791 3000 71.6% chr2 + 32394654 32394744 91 browser details YourSeq 37 2738 2791 3000 85.4% chr19 + 55292479 55292529 51 browser details YourSeq 35 2759 2799 3000 95.0% chr15 - 101208625 101208668 44 browser details YourSeq 35 2732 2797 3000 94.9% chr18 + 70539990 70540326 337 browser details YourSeq 35 2753 2791 3000 97.5% chr17 + 29030546 29031079 534 browser details YourSeq 35 2754 2794 3000 97.4% chr1 + 91249731 91249775 45 browser details YourSeq 34 2732 2791 3000 91.7% chr1 - 36673806 36673864 59 browser details YourSeq 33 2894 2961 3000 66.7% chr1 + 39489426 39489466 41 browser details YourSeq 31 2759 2794 3000 97.0% chr8 - 106206891 106206927 37 browser details YourSeq 31 2759 2794 3000 97.0% chr10 + 32756806 32756842 37 browser details YourSeq 30 2820 2851 3000 90.4% chr4 - 118151119 118151149 31 browser details YourSeq 30 2759 2791 3000 96.9% chr2 - 118073874 118073907 34 browser details YourSeq 30 543 575 3000 97.0% chr17 + 5291791 5291824 34 browser details YourSeq 30 2894 2960 3000 85.8% chr15 + 83078681 83078746 66 browser details YourSeq 30 2738 2791 3000 96.9% chr11 + 116462374 116462428 55 browser details YourSeq 29 2759 2794 3000 96.8% chr19 - 45972505 45972541 37

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: Ttyh2 tweety family member 2 [ Mus musculus (house mouse) ] Gene ID: 117160, updated on 12-Aug-2019

Gene summary

Official Symbol Ttyh2 provided by MGI Official Full Name tweety family member 2 provided by MGI Primary source MGI:MGI:2157091 See related Ensembl:ENSMUSG00000034714 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 1110001A03Rik Expression Broad expression in adrenal adult (RPKM 72.1), small intestine adult (RPKM 41.6) and 22 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 E2 See Ttyh2 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (114675468..114720984)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (114536782..114582298)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 2 transcripts

Gene: Ttyh2 ENSMUSG00000034714

Description tweety family member 2 [Source:MGI Symbol;Acc:MGI:2157091] Gene Synonyms 1110001A03Rik Location Chromosome 11: 114,675,431-114,720,977 forward strand. GRCm38:CM001004.2 About this gene This gene has 2 transcripts (splice variants), 250 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ttyh2-201 ENSMUST00000045779.5 3486 532aa ENSMUSP00000037821.5 Protein coding CCDS25608 Q3TH73 TSL:1 GENCODE basic APPRIS P1

Ttyh2-202 ENSMUST00000141111.1 695 No protein - lncRNA - - TSL:2

65.55 kb Forward strand 114.68Mb 114.70Mb 114.72Mb Genes Rpl38-204 >protein coding Dnaic2-201 >protein coding (Comprehensive set...

Rpl38-201 >protein coding Dnaic2-202 >protein coding

Rpl38-203 >protein coding Dnaic2-207 >lncRNA

Rpl38-202 >protein coding Dnaic2-204 >lncRNA

Ttyh2-201 >protein coding

Ttyh2-202 >lncRNA

Contigs AL645484.14 > AL663079.9 > Regulatory Build

114.68Mb 114.70Mb 114.72Mb Reverse strand 65.55 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000045779

45.55 kb Forward strand

Ttyh2-201 >protein coding

ENSMUSP00000037... Transmembrane heli... Low complexity (Seg) Pfam Tweety PANTHER Tweety

PTHR12424:SF6 CDD Tweety

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 532

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

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