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Mouse Tbc1d24 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tbc1d24 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tbc1d24 gene (NCBI Reference Sequence: NM_001163850 ; Ensembl: ENSMUSG00000036473 ) is located on Mouse chromosome 17. 8 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000168410). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tbc1d24 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-62L15 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 heterozygous for a knock-out allele show altered primary neuron maturation and survival, impaired endocytosis and an enlarged endosomal compartment in neurons, and a decrease in spontaneous neurotransmission.

Exon 3~4 covers 67.51% of the coding region. Start codon is in exon 3, and stop codon is in exon 8. The size of intron 2 for 5'-loxP site insertion: 3742 bp, and the size of intron 4 for 3'-loxP site insertion: 844 bp. The size of effective cKO region: ~2975 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 3 4 5 6 7 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tbc1d24 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(9475bp) | A(21.73% 2059) | C(25.89% 2453) | T(27.5% 2606) | G(24.88% 2357)

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% chr17 - 24186419 24189418 3000 browser details YourSeq 345 1849 2859 3000 91.0% chr15 - 88766259 88767366 1108 browser details YourSeq 307 1881 2626 3000 89.9% chr4 - 55393179 55393867 689 browser details YourSeq 292 1881 2508 3000 89.0% chr7 + 128568705 128569115 411 browser details YourSeq 281 1881 2508 3000 90.1% chr2 + 144576602 144577226 625 browser details YourSeq 273 1873 2508 3000 87.5% chr7 + 127762829 127763196 368 browser details YourSeq 271 1882 2508 3000 86.7% chr2 - 127002167 127002744 578 browser details YourSeq 269 1873 2508 3000 88.6% chr2 - 26859072 26859631 560 browser details YourSeq 269 1904 2510 3000 90.0% chr2 + 60162832 60163216 385 browser details YourSeq 268 1904 2656 3000 93.9% chr13 + 97004495 97005307 813 browser details YourSeq 259 1904 2508 3000 91.1% chr2 - 29878662 29879255 594 browser details YourSeq 259 1873 2508 3000 93.7% chr19 - 45985837 45986513 677 browser details YourSeq 247 1881 2510 3000 88.5% chr8 - 107027425 107027725 301 browser details YourSeq 244 2095 2677 3000 86.4% chr17 - 56301382 56301929 548 browser details YourSeq 242 1918 2530 3000 93.3% chr12 - 91797635 91798269 635 browser details YourSeq 241 2181 2873 3000 86.3% chr11 - 95307078 95307605 528 browser details YourSeq 240 2321 2641 3000 92.9% chr10 + 61397071 61397403 333 browser details YourSeq 230 1840 2833 3000 93.3% chr11 + 87419746 87420807 1062 browser details YourSeq 229 1965 2600 3000 90.4% chr1 + 136702123 136702719 597 browser details YourSeq 228 2321 2873 3000 88.0% chr4 + 155372983 155373455 473

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% chr17 - 24180444 24183443 3000 browser details YourSeq 23 1000 1022 3000 100.0% chr8 + 76565594 76565616 23 browser details YourSeq 22 1000 1021 3000 100.0% chr7 + 106030275 106030296 22 browser details YourSeq 22 1844 1865 3000 100.0% chr5 + 127447220 127447241 22 browser details YourSeq 22 1072 1095 3000 87.0% chr12 + 38141285 38141307 23 browser details YourSeq 21 591 611 3000 100.0% chr9 + 23315221 23315241 21 browser details YourSeq 21 1712 1732 3000 100.0% chr8 + 71581421 71581441 21 browser details YourSeq 20 2892 2917 3000 88.5% chr5 + 104110041 104110066 26

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: Tbc1d24 TBC1 domain family, member 24 [ Mus musculus (house mouse) ] Gene ID: 224617, updated on 10-Oct-2019

Gene summary

Official Symbol Tbc1d24 provided by MGI Official Full Name TBC1 domain family, member 24 provided by MGI Primary source MGI:MGI:2443456 See related Ensembl:ENSMUSG00000036473 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 mKIAA1171; 9630033P11; C530046L02Rik Expression Ubiquitous expression in CNS E18 (RPKM 17.3), whole brain E14.5 (RPKM 14.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 17; 17 A3.3 See Tbc1d24 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (24175431..24205562, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (24312376..24342507, complement)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 15 transcripts

Gene: Tbc1d24 ENSMUSG00000036473

Description TBC1 domain family, member 24 [Source:MGI Symbol;Acc:MGI:2443456] Gene Synonyms C530046L02Rik Location Chromosome 17: 24,175,431-24,205,562 reverse strand. GRCm38:CM001010.2 About this gene This gene has 15 transcripts (splice variants), 203 orthologues, 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

Tbc1d24- ENSMUST00000168410.8 7683 555aa ENSMUSP00000128868.2 Protein coding CCDS28477 Q3UUG6 TSL:1 205 GENCODE basic APPRIS P3

Tbc1d24- ENSMUST00000097376.9 7265 561aa ENSMUSP00000094989.3 Protein coding CCDS50012 Q3UUG6 TSL:1 202 GENCODE basic APPRIS ALT2

Tbc1d24- ENSMUST00000040474.10 6269 555aa ENSMUSP00000036458.7 Protein coding CCDS28477 Q3UUG6 TSL:1 201 GENCODE basic APPRIS P3

Tbc1d24- ENSMUST00000171189.7 4592 555aa ENSMUSP00000128001.1 Protein coding CCDS28477 Q3UUG6 TSL:1 206 GENCODE basic APPRIS P3

Tbc1d24- ENSMUST00000167791.8 4383 561aa ENSMUSP00000127005.2 Protein coding CCDS50012 Q3UUG6 TSL:1 203 GENCODE basic APPRIS ALT2

Tbc1d24- ENSMUST00000168378.7 4350 555aa ENSMUSP00000126107.1 Protein coding CCDS28477 Q3UUG6 TSL:1 204 GENCODE basic APPRIS P3

Tbc1d24- ENSMUST00000201960.3 4305 555aa ENSMUSP00000144208.1 Protein coding CCDS28477 Q3UUG6 TSL:5 213 GENCODE basic APPRIS P3

Tbc1d24- ENSMUST00000201301.3 4261 561aa ENSMUSP00000143949.1 Protein coding CCDS50012 Q3UUG6 TSL:5 208 GENCODE basic APPRIS ALT2

Tbc1d24- ENSMUST00000202925.3 4216 555aa ENSMUSP00000144575.1 Protein coding CCDS28477 Q3UUG6 TSL:5 215 GENCODE basic APPRIS P3

Tbc1d24- ENSMUST00000201089.3 4081 555aa ENSMUSP00000144250.1 Protein coding CCDS28477 Q3UUG6 TSL:1 207 GENCODE basic APPRIS P3

Tbc1d24- ENSMUST00000201805.3 3806 561aa ENSMUSP00000143883.1 Protein coding CCDS50012 Q3UUG6 TSL:1 212 GENCODE basic APPRIS

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ALT2

Tbc1d24- ENSMUST00000201583.1 893 182aa ENSMUSP00000144097.1 Protein coding - A0A0J9YUB2 TSL:5 210 GENCODE basic

Tbc1d24- ENSMUST00000201359.3 4098 325aa ENSMUSP00000144026.1 Nonsense mediated - Q8BRB6 TSL:1 209 decay

Tbc1d24- ENSMUST00000201716.1 434 No - lncRNA - - TSL:2 211 protein

Tbc1d24- ENSMUST00000202018.1 370 No - lncRNA - - TSL:3 214 protein

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50.13 kb Forward strand

24.17Mb 24.18Mb 24.19Mb 24.20Mb 24.21Mb Genes BC028777-203 >lncRNA (Comprehensive set...

BC028777-202 >lncRNA

BC028777-201 >lncRNA

Contigs < CT010502.14 AC166573.2 > < AC117577.16

Genes < Atp6v0c-201protein coding < Tbc1d24-205protein coding < Ntn3-203retained intron (Comprehensive set...

< Atp6v0c-205lncRNA < Tbc1d24-202protein coding < Ntn3-204lncRNA

< Atp6v0c-202protein coding < Tbc1d24-201protein coding < Tedc2-203retained intron

< Atp6v0c-203protein coding < Tbc1d24-207protein coding < Tedc2-201protein coding

< Atp6v0c-206lncRNA < Tbc1d24-203protein coding

< Atp6v0c-204lncRNA < Tbc1d24-212protein coding

< Tbc1d24-206protein coding < Ntn3-201protein coding

< Tbc1d24-208protein coding

< Tbc1d24-215protein coding

< Tbc1d24-213protein coding

< Tbc1d24-204protein coding < Ntn3-202nonsense mediated decay

< Tbc1d24-209nonsense mediated decay

< Tbc1d24-214lncRNA < Tbc1d24-211lncRNA

< Tbc1d24-210protein coding

< Tedc2-202nonsense mediated decay

Regulatory Build

24.17Mb 24.18Mb 24.19Mb 24.20Mb 24.21Mb Reverse strand 50.13 kb

Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000168410

< Tbc1d24-205protein coding

Reverse strand 30.11 kb

ENSMUSP00000128... Low complexity (Seg) Superfamily Rab-GTPase-TBC domain superfamily SMART Rab-GTPase-TBC domain TLDc domain

Pfam Rab-GTPase-TBC domain TLDc domain

PANTHER PTHR23353

TBC1 domain family member 24 Gene3D 1.10.472.80

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 555

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

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