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Mouse Tubgcp3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tubgcp3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tubgcp3 gene (NCBI Reference Sequence: NM_198031 ; Ensembl: ENSMUSG00000000759 ) is located on Mouse chromosome 8. 22 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 22 (Transcript: ENSMUST00000000776). 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 Tubgcp3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-125D20 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 6.81% 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: 2564 bp, and the size of intron 3 for 3'-loxP site insertion: 2922 bp. The size of effective cKO region: ~568 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 22 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tubgcp3 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(7068bp) | A(22.84% 1614) | C(21.97% 1553) | T(30.49% 2155) | G(24.7% 1746)

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% chr8 - 12661618 12664617 3000 browser details YourSeq 80 796 2826 3000 89.9% chr19 - 32119840 32535727 415888 browser details YourSeq 73 756 882 3000 93.1% chr10 - 13450920 13451245 326 browser details YourSeq 66 754 905 3000 91.1% chr16 + 96305335 96305860 526 browser details YourSeq 66 2742 2858 3000 79.8% chr11 + 74898740 74898836 97 browser details YourSeq 62 764 882 3000 88.7% chr17 - 30725523 30725643 121 browser details YourSeq 54 754 843 3000 93.9% chr1 - 38438069 38438396 328 browser details YourSeq 54 2779 2858 3000 83.8% chr13 + 44995291 44995370 80 browser details YourSeq 53 2752 2828 3000 95.0% chr12 - 51781306 51781395 90 browser details YourSeq 51 2778 2840 3000 90.5% chr7 - 46167891 46167953 63 browser details YourSeq 51 2778 2857 3000 80.9% chr6 + 141373814 141373891 78 browser details YourSeq 51 800 963 3000 88.8% chr11 + 25256456 25256703 248 browser details YourSeq 50 2778 2835 3000 93.2% chr14 - 34215305 34215362 58 browser details YourSeq 50 754 843 3000 90.0% chr10 - 59024444 59024591 148 browser details YourSeq 47 2780 2860 3000 86.2% chr11 - 76777019 76777109 91 browser details YourSeq 47 2778 2841 3000 92.8% chr4 + 9020532 9020596 65 browser details YourSeq 47 2784 2834 3000 96.1% chr10 + 79828312 79828362 51 browser details YourSeq 47 844 932 3000 78.4% chr10 + 63388303 63388381 79 browser details YourSeq 46 868 960 3000 94.4% chr11 + 95030220 95030465 246 browser details YourSeq 46 818 878 3000 96.2% chr10 + 67409409 67409760 352

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% chr8 - 12658050 12661049 3000 browser details YourSeq 72 2273 2382 3000 83.4% chr11 + 50865475 50865738 264 browser details YourSeq 70 2155 2367 3000 94.0% chr10 - 41325561 41326015 455 browser details YourSeq 68 2273 2369 3000 85.6% chr10 - 43803676 43803773 98 browser details YourSeq 66 2273 2382 3000 75.8% chr1 - 164006432 164006534 103 browser details YourSeq 65 2286 2364 3000 91.2% chr11 - 96672562 96672640 79 browser details YourSeq 65 2274 2353 3000 94.6% chr12 + 70848261 70848697 437 browser details YourSeq 65 2273 2364 3000 93.3% chr11 + 70285987 70286080 94 browser details YourSeq 65 2286 2360 3000 93.4% chr1 + 154069412 154069486 75 browser details YourSeq 64 2286 2380 3000 79.8% chr10 + 7696093 7696180 88 browser details YourSeq 63 2286 2364 3000 95.7% chr11 - 52897193 52897272 80 browser details YourSeq 63 2272 2353 3000 88.9% chr11 - 5409956 5410035 80 browser details YourSeq 63 2255 2353 3000 88.9% chr1 - 177219664 177219770 107 browser details YourSeq 63 2290 2366 3000 92.0% chr10 + 61344607 61387941 43335 browser details YourSeq 62 2286 2353 3000 95.6% chr9 - 63764285 63764352 68 browser details YourSeq 62 2273 2353 3000 89.2% chr12 - 25995011 25995090 80 browser details YourSeq 61 2286 2370 3000 82.3% chr11 - 50303682 50303763 82 browser details YourSeq 60 2286 2369 3000 94.3% chr12 - 22062461 22062549 89 browser details YourSeq 59 2286 2366 3000 87.4% chr11 - 14605640 14605718 79 browser details YourSeq 59 2286 2352 3000 94.1% chr10 + 60920350 60920416 67

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: Tubgcp3 tubulin, gamma complex associated protein 3 [ Mus musculus (house mouse) ] Gene ID: 259279, updated on 12-Aug-2019

Gene summary

Official Symbol Tubgcp3 provided by MGI Official Full Name tubulin, gamma complex associated protein 3 provided by MGI Primary source MGI:MGI:2183752 See related Ensembl:ENSMUSG00000000759 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as GCP3; Spc98p Expression Ubiquitous expression in thymus adult (RPKM 15.7), liver E14.5 (RPKM 14.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 8; 8 A1.1 See Tubgcp3 in Genome Data Viewer

Exon count: 23

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (12614277..12672854, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (12614277..12672100, complement)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 9 transcripts

Gene: Tubgcp3 ENSMUSG00000000759

Description tubulin, gamma complex associated protein 3 [Source:MGI Symbol;Acc:MGI:2183752] Gene Synonyms GCP3, Spc98p Location Chromosome 8: 12,614,277-12,672,248 reverse strand. GRCm38:CM001001.2 About this gene This gene has 9 transcripts (splice variants), 199 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tubgcp3- ENSMUST00000000776.14 3797 905aa ENSMUSP00000000776.8 Protein coding CCDS22103 P58854 TSL:1 201 GENCODE basic APPRIS P1

Tubgcp3- ENSMUST00000164774.7 3515 364aa ENSMUSP00000127741.1 Nonsense mediated - E9Q0R3 TSL:1 203 decay

Tubgcp3- ENSMUST00000165321.1 458 86aa ENSMUSP00000131051.1 Nonsense mediated - F7AJZ3 CDS 5' 206 decay incomplete TSL:3

Tubgcp3- ENSMUST00000172056.1 419 70aa ENSMUSP00000130106.1 Nonsense mediated - F6QMZ4 CDS 5' 209 decay incomplete TSL:5

Tubgcp3- ENSMUST00000167547.7 5724 No - Retained intron - - TSL:5 207 protein

Tubgcp3- ENSMUST00000164971.7 2977 No - Retained intron - - TSL:1 205 protein

Tubgcp3- ENSMUST00000168657.7 2018 No - Retained intron - - TSL:1 208 protein

Tubgcp3- ENSMUST00000164611.7 1178 No - Retained intron - - TSL:5 202 protein

Tubgcp3- ENSMUST00000164861.1 654 No - Retained intron - - TSL:2 204 protein

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77.97 kb Forward strand

Genes Gm26483-201 >miRNA (Comprehensive set...

4931415C17Rik-201 >lncRNA

4931415C17Rik-202 >lncRNA

Contigs < AC161505.4 < AC127677.4 Genes (Comprehensive set... < Tubgcp3-201protein coding

< Tubgcp3-203nonsense mediated decay

< Tubgcp3-208retained intron < Tubgcp3-204retained intron

< Tubgcp3-202retained intron < Tubgcp3-206nonsense mediated decay

< Tubgcp3-205retained intron

< Tubgcp3-207retained intron

< Tubgcp3-209nonsense mediated decay

Regulatory Build

Reverse strand 77.97 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000000776

< Tubgcp3-201protein coding

Reverse strand 57.97 kb

ENSMUSP00000000... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Gamma tubulin complex component protein, N-terminal

Gamma tubulin complex component, C-terminal PANTHER Gamma-tubulin complex component protein

PTHR19302:SF14

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

Variant Legend stop gained missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 905

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

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