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Mouse Trp53i11 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Trp53i11 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Trp53i11 gene (NCBI Reference Sequence: NM_001025246 ; Ensembl: ENSMUSG00000068735 ) is located on Mouse chromosome 2. 8 exons are identified, with the ATG start codon in exon 3 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000090554). Exon 4~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Trp53i11 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-151G14 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 4 starts from about 22.93% of the coding region. The knockout of Exon 4~6 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 514 bp, and the size of intron 6 for 3'-loxP site insertion: 365 bp. The size of effective cKO region: ~1161 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 4 5 6 7 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Trp53i11 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(7593bp) | A(20.64% 1567) | C(26.34% 2000) | T(24.18% 1836) | G(28.84% 2190)

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% chr2 + 93195014 93198013 3000 browser details YourSeq 30 2412 2464 3000 89.5% chr1 - 184876504 184876944 441 browser details YourSeq 26 2437 2466 3000 93.4% chr17 + 27740884 27740913 30 browser details YourSeq 24 2441 2466 3000 96.2% chr15 - 25712656 25712681 26 browser details YourSeq 24 2437 2464 3000 92.9% chr9 + 20868446 20868473 28 browser details YourSeq 24 2438 2465 3000 92.9% chr6 + 146195491 146195518 28 browser details YourSeq 23 1713 1738 3000 96.2% chr8 - 87325897 87325924 28 browser details YourSeq 23 1808 1833 3000 96.2% chr5 - 52110310 52110336 27 browser details YourSeq 23 2437 2465 3000 89.7% chr15 - 101045429 101045457 29 browser details YourSeq 23 1439 1465 3000 96.0% chr2 + 128443443 128443471 29 browser details YourSeq 22 2443 2464 3000 100.0% chr9 - 57275486 57275507 22 browser details YourSeq 22 2437 2464 3000 89.3% chr19 - 29509680 29509707 28 browser details YourSeq 21 388 408 3000 100.0% chr6 - 91601691 91601711 21 browser details YourSeq 21 2438 2464 3000 88.9% chr4 - 130004840 130004866 27 browser details YourSeq 21 2438 2464 3000 88.9% chr17 + 24651186 24651212 27 browser details YourSeq 20 2437 2462 3000 88.5% chr7 - 98179426 98179451 26 browser details YourSeq 20 1456 1485 3000 83.4% chr1 - 12985321 12985350 30

Note: The 3000 bp section upstream of Exon 4 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% chr2 + 93199175 93202174 3000 browser details YourSeq 56 2727 2782 3000 100.0% chr12 - 12667056 12667111 56 browser details YourSeq 45 2733 2782 3000 98.0% chr19 + 28277769 28277844 76 browser details YourSeq 29 2541 2569 3000 100.0% chr6 - 80432359 80432387 29 browser details YourSeq 29 2541 2569 3000 100.0% chr2 + 6876115 6876143 29 browser details YourSeq 25 1142 1168 3000 96.3% chr5 - 72497219 72497245 27 browser details YourSeq 25 2757 2781 3000 100.0% chr2 - 30504990 30505014 25 browser details YourSeq 24 1020 1044 3000 100.0% chr5 + 53324876 53324901 26 browser details YourSeq 23 2279 2301 3000 100.0% chr15 - 79787690 79787712 23 browser details YourSeq 22 926 947 3000 100.0% chr4 - 141628210 141628231 22 browser details YourSeq 22 1087 1108 3000 100.0% chr13 + 88241600 88241621 22 browser details YourSeq 22 2425 2446 3000 100.0% chr10 + 76225241 76225262 22 browser details YourSeq 21 2424 2444 3000 100.0% chr5 - 112206466 112206486 21 browser details YourSeq 21 1536 1556 3000 100.0% chr2 - 167842576 167842596 21 browser details YourSeq 21 982 1002 3000 100.0% chr14 - 25875536 25875556 21 browser details YourSeq 21 1517 1537 3000 100.0% chrX + 98436006 98436026 21 browser details YourSeq 21 2423 2443 3000 100.0% chr11 + 73342361 73342381 21

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Trp53i11 transformation related protein 53 inducible protein 11 [ Mus musculus (house mouse) ] Gene ID: 277414, updated on 24-Oct-2019

Gene summary

Official Symbol Trp53i11 provided by MGI Official Full Name transformation related protein 53 inducible protein 11 provided by MGI Primary source MGI:MGI:2670995 See related Ensembl:ENSMUSG00000068735 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 Tp53i11 Expression Broad expression in thymus adult (RPKM 53.1), colon adult (RPKM 33.8) and 24 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 E1 See Trp53i11 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (93187349..93201757)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (93027741..93041914)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 8 transcripts

Gene: Trp53i11 ENSMUSG00000068735

Description transformation related protein 53 inducible protein 11 [Source:MGI Symbol;Acc:MGI:2670995] Gene Synonyms Tp53i11 Location Chromosome 2: 93,187,548-93,201,759 forward strand. GRCm38:CM000995.2 About this gene This gene has 8 transcripts (splice variants), 204 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Trp53i11-202 ENSMUST00000111266.7 2703 189aa ENSMUSP00000106897.1 Protein coding CCDS16452 Q4QQM4 TSL:1 GENCODE basic APPRIS P1

Trp53i11-201 ENSMUST00000090554.10 2654 189aa ENSMUSP00000088042.4 Protein coding CCDS16452 Q4QQM4 TSL:1 GENCODE basic APPRIS P1

Trp53i11-208 ENSMUST00000150462.7 690 87aa ENSMUSP00000115918.1 Protein coding - A2AGS6 CDS 3' incomplete TSL:5

Trp53i11-203 ENSMUST00000127173.1 2899 No protein - lncRNA - - TSL:1

Trp53i11-205 ENSMUST00000140172.7 1752 No protein - lncRNA - - TSL:5

Trp53i11-207 ENSMUST00000147377.7 990 No protein - lncRNA - - TSL:5

Trp53i11-206 ENSMUST00000143164.1 952 No protein - lncRNA - - TSL:2

Trp53i11-204 ENSMUST00000132182.1 552 No protein - lncRNA - - TSL:3

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34.21 kb Forward strand

93.18Mb 93.19Mb 93.20Mb 93.21Mb Genes (Comprehensive set... Trp53i11-208 >protein coding

Trp53i11-202 >protein coding

Trp53i11-207 >lncRNA

Trp53i11-205 >lncRNA

Trp53i11-201 >protein coding

Trp53i11-204 >lncRNA Trp53i11-206 >lncRNA

Trp53i11-203 >lncRNA

Contigs AL683882.8 > AL691472.6 > Genes < Tspan18-202protein coding (Comprehensive set...

< Tspan18-201protein coding

Regulatory Build

93.18Mb 93.19Mb 93.20Mb 93.21Mb Reverse strand 34.21 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000090554

14.17 kb Forward strand

Trp53i11-201 >protein coding

ENSMUSP00000088... Transmembrane heli... Low complexity (Seg) Pfam Tumor protein p53-inducible protein 11 PANTHER Tumor protein p53-inducible protein 11

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 189

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

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