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Mouse Tnip1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tnip1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tnip1 gene (NCBI Reference Sequence: NM_001199275 ; Ensembl: ENSMUSG00000020400 ) is located on Mouse chromosome 11. 19 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 19 (Transcript: ENSMUST00000102731). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tnip1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-357E11 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 null allele exhibit perinatal lethality associated with anemia and focal apoptosis in the fetal liver. Mice homozygous for a gene trap allele exhibit partial prenatal lethality and SLE-like inflammatory disease.

Exon 5 starts from about 14.17% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1532 bp, and the size of intron 6 for 3'-loxP site insertion: 2280 bp. The size of effective cKO region: ~2504 bp.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 19 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tnip1 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(8605bp) | A(21.84% 1879) | C(25.85% 2224) | T(25.57% 2200) | G(26.75% 2302)

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% chr11 - 54938316 54941315 3000 browser details YourSeq 138 2442 2606 3000 97.4% chr2 + 102209051 102209217 167 browser details YourSeq 136 2458 2607 3000 96.7% chr2 - 11525582 11572650 47069 browser details YourSeq 130 2442 2607 3000 90.5% chr18 - 60841279 60841440 162 browser details YourSeq 129 2443 2607 3000 94.5% chr2 - 151003187 151003540 354 browser details YourSeq 129 2445 2607 3000 91.2% chrX + 61911690 61911847 158 browser details YourSeq 128 2442 2607 3000 88.0% chr5 - 136390113 136390256 144 browser details YourSeq 127 2448 2607 3000 86.9% chr14 + 15915088 15915233 146 browser details YourSeq 126 2470 2618 3000 96.4% chr2 + 104747573 104747970 398 browser details YourSeq 126 2460 2607 3000 95.2% chr18 + 13993359 13993519 161 browser details YourSeq 125 2460 2607 3000 93.0% chr6 + 3997068 3997214 147 browser details YourSeq 123 2040 2607 3000 81.0% chr12 - 70139784 70139961 178 browser details YourSeq 123 2445 2606 3000 86.5% chr6 + 18854389 18854530 142 browser details YourSeq 121 2442 2594 3000 88.3% chr5 - 55475438 55475574 137 browser details YourSeq 120 2460 2606 3000 93.1% chr1 - 110354696 110354847 152 browser details YourSeq 119 2414 2599 3000 96.1% chr9 - 20896596 20896843 248 browser details YourSeq 119 2471 2607 3000 95.5% chr15 - 8053780 8053926 147 browser details YourSeq 116 2452 2597 3000 95.4% chr7 - 63487269 63487603 335 browser details YourSeq 116 2457 2607 3000 87.0% chr2 - 31870189 31870318 130 browser details YourSeq 115 2479 2607 3000 96.8% chr1 + 156508947 156509080 134

Note: The 3000 bp section upstream of Exon 5 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% chr11 - 54933211 54936210 3000 browser details YourSeq 207 2530 2963 3000 95.3% chr7 + 122935966 122936623 658 browser details YourSeq 197 2515 2966 3000 92.0% chr5 - 67852052 67852627 576 browser details YourSeq 195 2698 2969 3000 94.5% chrX + 103974512 103975145 634 browser details YourSeq 193 2646 2970 3000 95.3% chr15 - 102311037 102311704 668 browser details YourSeq 192 2551 2966 3000 95.7% chr10 - 121478433 121479069 637 browser details YourSeq 184 2596 2969 3000 88.4% chr8 - 110484612 110484906 295 browser details YourSeq 183 2714 2969 3000 96.5% chr6 - 88475168 88475591 424 browser details YourSeq 183 2764 2972 3000 97.0% chr2 - 144765896 144766111 216 browser details YourSeq 183 2773 2976 3000 95.5% chr3 + 95592563 95592774 212 browser details YourSeq 183 2766 2969 3000 93.5% chr12 + 4084866 4085066 201 browser details YourSeq 181 2765 2969 3000 94.5% chr9 - 95794713 95794916 204 browser details YourSeq 181 2774 2969 3000 94.3% chr11 + 115504479 115504670 192 browser details YourSeq 179 2767 2969 3000 95.9% chr14 - 55696286 55696489 204 browser details YourSeq 179 2774 2970 3000 93.3% chr3 + 30622436 30622628 193 browser details YourSeq 179 2780 2969 3000 97.4% chr13 + 66989444 66989635 192 browser details YourSeq 178 2767 2970 3000 96.4% chr10 - 128260329 128260539 211 browser details YourSeq 178 2765 2970 3000 95.5% chr5 + 108047753 108047998 246 browser details YourSeq 177 2779 2969 3000 94.7% chr4 - 33211536 33211723 188 browser details YourSeq 177 2768 2968 3000 95.4% chr11 - 103965861 103966063 203

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: Tnip1 TNFAIP3 interacting protein 1 [ Mus musculus (house mouse) ] Gene ID: 57783, updated on 12-Aug-2019

Gene summary

Official Symbol Tnip1 provided by MGI Official Full Name TNFAIP3 interacting protein 1 provided by MGI Primary source MGI:MGI:1926194 See related Ensembl:ENSMUSG00000020400 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 Nef; VAN; ABIN; Naf1; ABIN1; ABIN-1; AU018810 Expression Ubiquitous expression in colon adult (RPKM 26.8), duodenum adult (RPKM 23.7) and 28 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 B1.3 See Tnip1 in Genome Data Viewer

Exon count: 20

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (54910787..54962956, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (54724289..54776440, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 12 transcripts

Gene: Tnip1 ENSMUSG00000020400

Description TNFAIP3 interacting protein 1 [Source:MGI Symbol;Acc:MGI:1926194] Gene Synonyms A20-binding inhibitor of NF-kappa B activation, ABIN1, Nef, VAN Location Chromosome 11: 54,910,785-54,962,917 reverse strand. GRCm38:CM001004.2 About this gene This gene has 12 transcripts (splice variants), 200 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 68 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tnip1-203 ENSMUST00000102731.7 2973 647aa ENSMUSP00000099792.1 Protein coding CCDS24704 E9QM75 TSL:5 GENCODE basic APPRIS P3

Tnip1-206 ENSMUST00000108889.9 2834 647aa ENSMUSP00000104517.3 Protein coding CCDS24704 E9QM75 TSL:1 GENCODE basic APPRIS P3

Tnip1-201 ENSMUST00000018482.12 2810 647aa ENSMUSP00000018482.6 Protein coding CCDS24704 E9QM75 TSL:1 GENCODE basic APPRIS P3

Tnip1-202 ENSMUST00000102730.8 2787 646aa ENSMUSP00000099791.2 Protein coding CCDS70188 D3Z2W0 TSL:1 GENCODE basic APPRIS ALT2

Tnip1-204 ENSMUST00000108885.7 2686 594aa ENSMUSP00000104513.1 Protein coding CCDS56769 Q9WUU8 TSL:1 GENCODE basic APPRIS ALT2

Tnip1-205 ENSMUST00000108886.7 2642 594aa ENSMUSP00000104514.1 Protein coding CCDS56769 Q9WUU8 TSL:1 GENCODE basic APPRIS ALT2

Tnip1-208 ENSMUST00000126703.7 941 272aa ENSMUSP00000122836.1 Protein coding - D3Z3B6 CDS 3' incomplete TSL:5

Tnip1-212 ENSMUST00000155316.1 878 214aa ENSMUSP00000116721.1 Protein coding - D3Z410 CDS 3' incomplete TSL:5

Tnip1-209 ENSMUST00000132224.1 442 No protein - Retained intron - - TSL:3

Tnip1-207 ENSMUST00000124304.1 1786 No protein - lncRNA - - TSL:1

Tnip1-211 ENSMUST00000151736.1 391 No protein - lncRNA - - TSL:2

Tnip1-210 ENSMUST00000132776.1 347 No protein - lncRNA - - TSL:3

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72.13 kb Forward strand

Genes Gpx3-202 >protein coding Gm12230-201 >processed pseudogene (Comprehensive set...

Gpx3-201 >protein coding

Gpx3-203 >protein coding

Contigs AL593846.15 > Genes (Comprehensive set... < Tnip1-206protein coding

< Tnip1-203protein coding

< Tnip1-205protein coding

< Tnip1-201protein coding

< Tnip1-202protein coding

< Tnip1-207lncRNA

< Tnip1-204protein coding

< Tnip1-209retained intron < Tnip1-212protein coding

< Tnip1-208protein coding

< Tnip1-211lncRNA

< Tnip1-210lncRNA

Regulatory Build

Reverse strand 72.13 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

pseudogene processed transcript RNA gene

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Transcript: ENSMUST00000102731

< Tnip1-203protein coding

Reverse strand 52.13 kb

ENSMUSP00000099... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) PANTHER TNFAIP3-interacting protein 1

PTHR31882 Gene3D 1.20.5.990

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

Variant Legend frameshift variant inframe deletion missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 647

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

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