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Mouse Tusc3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tusc3 conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tusc3 gene (NCBI Reference Sequence: NM_030254 ; Ensembl: ENSMUSG00000039530 ) is located on Mouse chromosome 8. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000167992). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tusc3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-34J18 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 2 starts from about 13.35% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 4042 bp, and the size of intron 2 for 3'-loxP site insertion: 418 bp. The size of effective cKO region: ~629 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 2 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tusc3 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(7088bp) | A(25.35% 1797) | C(21.44% 1520) | T(33.99% 2409) | G(19.22% 1362)

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 + 39043266 39046265 3000 browser details YourSeq 131 1884 2136 3000 91.1% chr17 - 87374290 87374729 440 browser details YourSeq 84 2044 2185 3000 90.0% chr17 - 87374396 87374729 334 browser details YourSeq 83 1884 2040 3000 81.0% chr17 - 87374290 87374431 142 browser details YourSeq 72 1884 2183 3000 76.2% chr3 - 152108396 152108587 192 browser details YourSeq 45 2080 2152 3000 87.5% chr17 - 87374327 87374418 92 browser details YourSeq 41 2010 2096 3000 87.3% chr14 - 55262816 55262900 85 browser details YourSeq 41 1898 1952 3000 93.7% chr14 - 55262816 55262900 85 browser details YourSeq 41 1914 1968 3000 93.7% chr14 - 55262816 55262900 85 browser details YourSeq 41 1930 1984 3000 93.7% chr14 - 55262816 55262900 85 browser details YourSeq 41 2026 2080 3000 93.7% chr14 - 55262816 55262900 85 browser details YourSeq 37 1882 1990 3000 60.5% chr12 - 70552454 70552496 43 browser details YourSeq 35 1962 2000 3000 97.4% chr14 - 55262816 55262900 85 browser details YourSeq 24 1882 1908 3000 96.2% chr14 - 55262859 55262900 42 browser details YourSeq 24 2168 2191 3000 100.0% chr8 + 39045145 39045168 24

Note: The 3000 bp section upstream of Exon 2 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 + 39046895 39049894 3000 browser details YourSeq 40 505 555 3000 95.6% chr2 + 27892696 27893039 344 browser details YourSeq 36 502 541 3000 97.5% chr1 + 184412001 184412180 180 browser details YourSeq 34 504 557 3000 75.7% chr13 + 91977958 91978003 46 browser details YourSeq 32 510 550 3000 92.4% chr11 + 114796923 114796977 55 browser details YourSeq 31 502 542 3000 97.1% chr17 - 43730670 43730766 97 browser details YourSeq 29 502 542 3000 96.9% chr10 - 121485076 121485126 51

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tusc3 tumor suppressor candidate 3 [ Mus musculus (house mouse) ] Gene ID: 80286, updated on 12-Aug-2019

Gene summary

Official Symbol Tusc3 provided by MGI Official Full Name tumor suppressor candidate 3 provided by MGI Primary source MGI:MGI:1933134 See related Ensembl:ENSMUSG00000039530 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 N33; AU022242; BC003311 Annotation Annotation category: suggests misassembly information Expression Orthologs Ubiquitous expression in CNS E18 (RPKM 43.6), whole brain E14.5 (RPKM 27.6) and 27 other tissues See more human all

Genomic context

Location: 8; 8 A4 See Tusc3 in Genome Data Viewer

Exon count: 14

Annotation Status Assembly Chr Location release

GRCm38.p6 108 current 8 NC_000074.6 (39005351..39167050) (GCF_000001635.26)

previous NC_000074.5 (40068921..40193932) , Build 37.2 MGSCv37 (GCF_000001635.18) 8 assembly (40242614..40250468)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 6 transcripts

Gene: Tusc3 ENSMUSG00000039530

Description tumor suppressor candidate 3 [Source:MGI Symbol;Acc:MGI:1933134] Location Chromosome 8: 39,005,845-39,165,114 forward strand. GRCm38:CM001001.2 About this gene This gene has 6 transcripts (splice variants), 202 orthologues, 1 paralogue, 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

Tusc3- ENSMUST00000167992.7 1511 347aa ENSMUSP00000126080.1 Protein coding CCDS85529 Q8BTV1 TSL:1 201 GENCODE basic APPRIS P1

Tusc3- ENSMUST00000209440.1 1535 347aa ENSMUSP00000148134.1 Protein coding - Q8BTV1 TSL:1 203 GENCODE basic APPRIS P1

Tusc3- ENSMUST00000169034.1 1262 105aa ENSMUSP00000129916.1 Protein coding - Q99J74 TSL:1 202 GENCODE basic

Tusc3- ENSMUST00000210890.1 588 196aa ENSMUSP00000147691.1 Protein coding - A0A1B0GRW4 CDS 5' and 3' 205 incomplete TSL:3

Tusc3- ENSMUST00000211241.1 1264 105aa ENSMUSP00000148022.1 Nonsense mediated - Q99J74 TSL:5 206 decay

Tusc3- ENSMUST00000209970.1 2667 No - Retained intron - - TSL:1 204 protein

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179.27 kb Forward strand 39.00Mb 39.05Mb 39.10Mb 39.15Mb Genes (Comprehensive set... Tusc3-203 >protein coding

Tusc3-201 >protein coding

Tusc3-202 >protein coding

Tusc3-206 >nonsense mediated decay

Tusc3-204 >retained intron

Tusc3-205 >protein coding

Contigs AC168314.2 > Regulatory Build

39.00Mb 39.05Mb 39.10Mb 39.15Mb Reverse strand 179.27 kb

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

Gene Legend Protein Coding

Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000167992

159.25 kb Forward strand

Tusc3-201 >protein coding

ENSMUSP00000126... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Thioredoxin-like superfamily

Pfam Oligosaccharyl transferase complex, subunit OST3/OST6

PANTHER PTHR12692:SF1

Oligosaccharyl transferase complex, subunit OST3/OST6 Gene3D 3.40.30.10

CDD cd02947

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 347

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

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