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Mouse Tusc2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tusc2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tusc2 gene (NCBI Reference Sequence: NM_019742 ; Ensembl: ENSMUSG00000010054 ) is located on Mouse chromosome 9. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000010198). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tusc2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-418E8 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 homozygous for a knock-out allele develop signs of autoimmune disease, such as vasculitis, glomerulonephritis, anemia and circulating autoantibodies, weigh less, and show impaired NK cell maturation, lower IL-15 production, a higher frequency of spontaneous vascular tumors, and premature death.

Exon 2~3 covers 55.76% of the coding region. Start codon is in exon 1, and stop codon is in exon 3. The size of intron 1 for 5'-loxP site insertion: 1043 bp. The size of effective cKO region: ~2287 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 2 3 1 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Tusc2 cKO region Exon of mouse Hyal2 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(6834bp) | A(24.7% 1688) | C(26.09% 1783) | T(23.35% 1596) | G(25.86% 1767)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr9 + 107561327 107564326 3000 browser details YourSeq 114 2056 2201 3000 89.1% chr4 + 49412302 49412447 146 browser details YourSeq 113 2056 2196 3000 90.1% chr4 + 49342243 49342383 141 browser details YourSeq 55 1395 1506 3000 95.2% chr1 + 131665515 131665644 130 browser details YourSeq 54 1450 1516 3000 91.1% chr14 - 54507449 54507518 70 browser details YourSeq 54 1450 1516 3000 92.2% chr7 + 145060595 145060671 77 browser details YourSeq 53 1395 1497 3000 95.0% chr5 - 67828073 67828191 119 browser details YourSeq 51 1356 1503 3000 94.7% chr17 + 48271733 48271886 154 browser details YourSeq 50 1391 1493 3000 89.1% chr1 - 37645861 37646396 536 browser details YourSeq 50 1472 1580 3000 94.7% chr17 + 88157911 88158155 245 browser details YourSeq 49 1395 1503 3000 93.0% chr6 - 30034340 30034844 505 browser details YourSeq 49 1386 1488 3000 98.2% chr15 - 76160172 76160603 432 browser details YourSeq 48 1457 1632 3000 85.0% chr9 - 101136728 101136896 169 browser details YourSeq 48 1395 1498 3000 94.6% chr3 - 145286441 145286561 121 browser details YourSeq 47 1395 1516 3000 88.4% chr11 - 101612343 101612474 132 browser details YourSeq 46 1432 1514 3000 71.3% chr6 + 115575363 115575436 74 browser details YourSeq 45 1457 1510 3000 94.2% chr4 - 47081211 47081265 55 browser details YourSeq 45 1453 1518 3000 80.0% chr13 - 104051816 104051874 59 browser details YourSeq 43 1461 1506 3000 97.8% chr5 - 123918288 123918337 50 browser details YourSeq 43 1457 1515 3000 82.4% chr2 - 131026474 131026527 54

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% chr9 + 107564911 107567910 3000 browser details YourSeq 999 13 1200 3000 93.4% chr4 + 49342585 49414292 71708 browser details YourSeq 649 10 896 3000 92.7% chr4 + 49412641 49413989 1349 browser details YourSeq 278 888 1200 3000 94.9% chr4 + 49343435 49343747 313 browser details YourSeq 37 1802 1839 3000 100.0% chr6 + 4219590 4219628 39 browser details YourSeq 35 1817 1871 3000 92.9% chr9 - 39073160 39073222 63 browser details YourSeq 29 1803 1839 3000 87.5% chr9 - 72333023 72333057 35 browser details YourSeq 21 1810 1830 3000 100.0% chr1 - 4680235 4680255 21 browser details YourSeq 21 1813 1835 3000 95.7% chr1 + 20387531 20387553 23 browser details YourSeq 20 1815 1834 3000 100.0% chr8 + 116149671 116149690 20

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: Tusc2 tumor suppressor 2, mitochondrial calcium regulator [ Mus musculus (house mouse) ] Gene ID: 80385, updated on 10-Oct-2019

Gene summary

Official Symbol Tusc2 provided by MGI Official Full Name tumor suppressor 2, mitochondrial calcium regulator provided by MGI Primary source MGI:MGI:1931086 See related Ensembl:ENSMUSG00000010054 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 PAP; Fus1; Lgcc; Pdap2; AA407686; 1190001E22Rik Expression Ubiquitous expression in adrenal adult (RPKM 49.1), colon adult (RPKM 39.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 F1 See Tusc2 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (107563255..107566108)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (107465586..107468439)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 3 transcripts

Gene: Tusc2 ENSMUSG00000010054

Description tumor suppressor 2, mitochondrial calcium regulator [Source:MGI Symbol;Acc:MGI:1931086] Gene Synonyms 1190001E22Rik, Fus1, Pdap2 Location Chromosome 9: 107,563,255-107,566,112 forward strand. GRCm38:CM001002.2 About this gene This gene has 3 transcripts (splice variants), 254 orthologues, is a member of 1 Ensembl protein family and is associated with 21 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tusc2-201 ENSMUST00000010198.4 1665 110aa ENSMUSP00000010198.3 Protein coding CCDS23495 Q542N4 Q9WVF8 TSL:1 GENCODE basic APPRIS P1

Tusc2-203 ENSMUST00000193418.1 410 102aa ENSMUSP00000141635.1 Protein coding - A0A0A6YWP5 TSL:3 GENCODE basic

Tusc2-202 ENSMUST00000193292.1 302 No protein - lncRNA - - TSL:3

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22.86 kb Forward strand 107.555Mb 107.560Mb 107.565Mb 107.570Mb 107.575Mb Genes (Comprehensive set... Rassf1-202 >protein coding Tusc2-201 >protein coding Hyal2-207 >protein coding

Rassf1-205 >retained intronRassf1-203 >lncRNA Tusc2-202 >lncRNA Hyal2-202 >protein codingHyal2-205 >retained intron

Rassf1-204 >protein coding Tusc2-203 >protein coding Hyal2-201 >protein coding

Rassf1-209 >nonsense mediated decay Hyal2-203 >protein coding

Rassf1-201 >protein coding Hyal2-204 >protein coding

Rassf1-210 >lncRNARassf1-206 >retained intron Hyal2-206 >protein coding

Rassf1-208 >retained intron

Rassf1-207 >retained intron

Contigs AL672219.7 > Genes < Gm9917-201lncRNA < Gm16343-201lncRNA (Comprehensive set...

< Gm9917-202lncRNA

< Gm9917-204lncRNA

< Gm9917-203lncRNA

Regulatory Build

107.555Mb 107.560Mb 107.565Mb 107.570Mb 107.575Mb Reverse strand 22.86 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000010198

2.86 kb Forward strand

Tusc2-201 >protein coding

ENSMUSP00000010... MobiDB lite Low complexity (Seg) Pfam Tumour suppressor candidate 2 PANTHER Tumour suppressor candidate 2

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

Variant Legend synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 100 110

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

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