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Mouse Ttc4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ttc4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ttc4 gene (NCBI Reference Sequence: NM_028209 ; Ensembl: ENSMUSG00000025413 ) is located on Mouse chromosome 4. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000026480). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ttc4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-170O23 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 5 starts from about 40.59% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2662 bp, and the size of intron 5 for 3'-loxP site insertion: 1088 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 5 6 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ttc4 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(7125bp) | A(26.37% 1879) | C(22.19% 1581) | T(27.21% 1939) | G(24.22% 1726)

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% chr4 - 106671999 106674998 3000 browser details YourSeq 278 1751 2092 3000 91.0% chr8 - 35165887 35166230 344 browser details YourSeq 267 1750 2089 3000 90.7% chr17 + 79546270 80001785 455516 browser details YourSeq 262 1754 2090 3000 90.0% chr9 + 45214721 45215060 340 browser details YourSeq 261 1751 2097 3000 90.2% chr17 - 29540884 29541229 346 browser details YourSeq 261 1751 2090 3000 89.5% chr9 + 60805427 60805769 343 browser details YourSeq 259 1751 2092 3000 87.8% chr18 + 9581644 9581979 336 browser details YourSeq 258 1750 2090 3000 87.6% chr13 - 53006668 53006990 323 browser details YourSeq 258 1751 2151 3000 87.8% chr6 + 4769693 4770106 414 browser details YourSeq 258 1753 2090 3000 88.5% chr4 + 153910374 153910713 340 browser details YourSeq 257 1753 2089 3000 88.9% chr16 - 95416511 95416849 339 browser details YourSeq 256 1750 2090 3000 88.8% chr14 + 65273025 65273368 344 browser details YourSeq 255 1755 2090 3000 89.1% chr4 - 151678723 151679061 339 browser details YourSeq 254 1750 2090 3000 87.7% chr15 - 83520339 83520682 344 browser details YourSeq 253 1750 2087 3000 89.4% chr11 + 49813141 49813473 333 browser details YourSeq 253 1751 2088 3000 90.0% chr1 + 190508539 190508889 351 browser details YourSeq 252 1713 2088 3000 91.3% chr11 - 43496493 43497146 654 browser details YourSeq 250 1750 2092 3000 86.9% chr2 - 131572220 131572564 345 browser details YourSeq 250 1750 2090 3000 88.9% chr2 + 170552987 170553333 347 browser details YourSeq 249 1751 2090 3000 89.9% chr10 - 120619770 120620111 342

Note: The 3000 bp section upstream of Exon 5 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% chr4 - 106668374 106671373 3000 browser details YourSeq 241 2293 2645 3000 86.3% chr4 + 123791160 123791508 349 browser details YourSeq 237 2277 2640 3000 85.5% chr12 - 17560312 17560674 363 browser details YourSeq 224 2257 2645 3000 89.0% chr8 - 124558615 124559001 387 browser details YourSeq 218 2252 2647 3000 85.5% chr15 - 64849740 64850133 394 browser details YourSeq 217 2252 2641 3000 87.1% chr17 + 15437989 15438376 388 browser details YourSeq 215 2286 2641 3000 85.6% chr9 - 88507841 88508196 356 browser details YourSeq 214 2252 2641 3000 88.0% chr1 + 20323840 20324226 387 browser details YourSeq 212 2252 2628 3000 86.1% chr17 + 16110000 16111863 1864 browser details YourSeq 211 2286 2647 3000 89.9% chrX - 80696423 80696784 362 browser details YourSeq 210 2286 2646 3000 88.5% chr12 - 19028420 19028779 360 browser details YourSeq 210 2283 2628 3000 87.6% chr13 + 53299241 53299598 358 browser details YourSeq 209 2286 2641 3000 88.6% chr4 - 112375079 112375432 354 browser details YourSeq 209 2286 2647 3000 88.2% chr4 - 112376559 112376919 361 browser details YourSeq 209 2286 2647 3000 87.3% chr17 + 6099745 6100107 363 browser details YourSeq 208 2286 2647 3000 89.8% chr2 - 180866527 180866888 362 browser details YourSeq 207 2286 2649 3000 84.7% chr9 - 17031732 17032095 364 browser details YourSeq 207 2182 2641 3000 85.8% chr1 - 116805866 116806419 554 browser details YourSeq 206 2252 2641 3000 87.4% chr9 + 41691654 41692041 388 browser details YourSeq 205 2286 2645 3000 89.0% chr15 - 99858577 99858938 362

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ttc4 tetratricopeptide repeat domain 4 [ Mus musculus (house mouse) ] Gene ID: 72354, updated on 10-Oct-2019

Gene summary

Official Symbol Ttc4 provided by MGI Official Full Name tetratricopeptide repeat domain 4 provided by MGI Primary source MGI:MGI:1919604 See related Ensembl:ENSMUSG00000025413 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 L62; AF007005; 2810002P21Rik Expression Ubiquitous expression in testis adult (RPKM 33.7), CNS E18 (RPKM 31.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 C7 See Ttc4 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (106656806..106679002, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (106334413..106351549, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 9 transcripts

Gene: Ttc4 ENSMUSG00000025413

Description tetratricopeptide repeat domain 4 [Source:MGI Symbol;Acc:MGI:1919604] Gene Synonyms 2810002P21Rik, L62 Location Chromosome 4: 106,662,256-106,678,944 reverse strand. GRCm38:CM000997.2 About this gene This gene has 9 transcripts (splice variants), 208 orthologues, 18 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ttc4-201 ENSMUST00000026480.12 2121 386aa ENSMUSP00000026480.6 Protein coding CCDS18424 Q8R3H9 TSL:1 GENCODE basic APPRIS P2

Ttc4-202 ENSMUST00000106772.9 935 286aa ENSMUSP00000102384.3 Protein coding - A2AVQ9 TSL:5 GENCODE basic APPRIS ALT2

Ttc4-207 ENSMUST00000135676.1 752 241aa ENSMUSP00000116620.1 Protein coding - A2AVR1 CDS 3' incomplete TSL:5

Ttc4-209 ENSMUST00000142342.7 2118 No protein - lncRNA - - TSL:1

Ttc4-206 ENSMUST00000132650.1 851 No protein - lncRNA - - TSL:3

Ttc4-203 ENSMUST00000127133.1 758 No protein - lncRNA - - TSL:2

Ttc4-205 ENSMUST00000132572.1 725 No protein - lncRNA - - TSL:2

Ttc4-208 ENSMUST00000139687.1 691 No protein - lncRNA - - TSL:2

Ttc4-204 ENSMUST00000127571.7 383 No protein - lncRNA - - TSL:5

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36.69 kb Forward strand

106.66Mb 106.67Mb 106.68Mb Genes Pars2-203 >protein coding (Comprehensive set...

Pars2-202 >protein coding

Pars2-204 >lncRNA

Pars2-201 >protein coding

Contigs AL929585.11 > Genes (Comprehensive set... < Ttc4-201protein coding < Mroh7-201protein coding

< Ttc4-209lncRNA < Mroh7-202lncRNA

< Ttc4-206lncRNA < Ttc4-203lncRNA < Ttc4-204lncRNA < Mroh7-203retained intron

< Ttc4-202protein coding < Mroh7-206lncRNA

< Ttc4-208lncRNA < Ttc4-205lncRNA

< Ttc4-207protein coding

Regulatory Build

106.66Mb 106.67Mb 106.68Mb Reverse strand 36.69 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000026480

< Ttc4-201protein coding

Reverse strand 16.69 kb

ENSMUSP00000026... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Tetratricopeptide-like helical domain superfamily

SMART Tetratricopeptide repeat PROSITE profiles Tetratricopeptide repeat-containing domain

PANTHER PTHR46035

PTHR46035:SF4 Gene3D Tetratricopeptide-like helical domain superfamily

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 386

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

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