Mouse Tnip2 Knockout Project (CRISPR/Cas9)

http://www.alphaknockout.com/ Mouse Tnip2 Knockout Project (CRISPR/Cas9)

Objective: To create a Tnip2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tnip2 gene (NCBI Reference Sequence: NM_139064 ; Ensembl: ENSMUSG00000059866 ) is located on Mouse chromosome 5. 6 exons are identified , with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000087737). Exon 3~5 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a null allele have impaired IL-1 response and macrophage physiology.

Exon 3 starts from about 44.26% of the coding region. Exon 3~5 covers 35.58% of the coding region. The size of effective KO region: ~1818 bp. The KO 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 3 4 5 6

Legends Exon of mouse Tnip2 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 5 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(24.65% 493) | C(21.6% 432) | G(26.4% 528) | T(27.35% 547)

Note: The 2000 bp section upstream of Exon 3 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(23.2% 464) | C(22.65% 453) | G(24.85% 497) | T(29.3% 586)

Note: The 2000 bp section downstream of Exon 5 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 2000 1 2000 2000 100.0% chr5 - 34500998 34502997 2000 browser details YourSeq 46 611 751 2000 87.1% chr13 - 26935381 26935527 147 browser details YourSeq 41 542 588 2000 95.6% chr17 + 83903052 83903101 50 browser details YourSeq 31 722 759 2000 97.0% chr14 + 79332495 79332534 40 browser details YourSeq 29 555 589 2000 93.8% chr3 + 129242831 129242865 35 browser details YourSeq 26 555 589 2000 88.3% chr15 - 53684693 53684731 39 browser details YourSeq 26 862 890 2000 96.6% chr6 + 107243116 107243146 31 browser details YourSeq 24 1823 1846 2000 100.0% chr15 - 21704518 21704541 24 browser details YourSeq 23 660 756 2000 61.9% chr16 - 89777601 89777697 97 browser details YourSeq 23 206 228 2000 100.0% chr13 - 30969314 30969336 23 browser details YourSeq 23 91 113 2000 100.0% chr1 + 36375109 36375131 23 browser details YourSeq 22 618 643 2000 92.4% chr2 + 37683429 37683454 26 browser details YourSeq 22 864 886 2000 100.0% chr10 + 78797897 78797921 25 browser details YourSeq 21 1821 1841 2000 100.0% chr13 - 100955556 100955576 21 browser details YourSeq 21 1821 1841 2000 100.0% chr13 - 24673356 24673376 21 browser details YourSeq 21 797 817 2000 100.0% chr11 - 76593081 76593101 21 browser details YourSeq 21 715 735 2000 100.0% chr9 + 26527626 26527646 21 browser details YourSeq 21 139 159 2000 100.0% chr2 + 140199626 140199646 21 browser details YourSeq 20 139 158 2000 100.0% chr1 - 34412080 34412099 20

Note: The 2000 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 2000 1 2000 2000 100.0% chr5 - 34497180 34499179 2000 browser details YourSeq 43 1202 1621 2000 59.6% chr5 + 76263672 76263842 171 browser details YourSeq 40 1141 1227 2000 97.8% chr19 - 8940141 8940241 101 browser details YourSeq 33 1194 1229 2000 97.3% chr15 + 42513416 42513452 37 browser details YourSeq 33 1193 1229 2000 97.3% chr14 + 54548260 54548298 39 browser details YourSeq 32 1196 1230 2000 97.1% chr9 + 103157104 103157139 36 browser details YourSeq 32 1192 1225 2000 100.0% chr17 + 61579186 61579224 39 browser details YourSeq 32 1196 1229 2000 100.0% chr12 + 70852297 70852332 36 browser details YourSeq 31 1195 1230 2000 94.3% chr18 - 41365619 41365656 38 browser details YourSeq 31 1195 1229 2000 91.0% chr18 - 28215170 28215203 34 browser details YourSeq 31 1191 1229 2000 91.5% chr16 + 52196071 52196126 56 browser details YourSeq 30 1195 1230 2000 81.9% chr17 - 57120511 57120543 33 browser details YourSeq 30 1195 1230 2000 94.2% chr13 - 17060186 17060224 39 browser details YourSeq 30 1192 1226 2000 94.0% chr15 + 94043472 94043506 35 browser details YourSeq 30 1195 1227 2000 87.1% chr15 + 75303812 75303842 31 browser details YourSeq 30 1194 1226 2000 87.1% chr1 + 17434941 17434971 31 browser details YourSeq 29 1195 1227 2000 90.4% chrX - 45521396 45521427 32 browser details YourSeq 29 1198 1229 2000 96.8% chr7 - 57251061 57251092 32 browser details YourSeq 29 1202 1231 2000 100.0% chr3 - 115400763 115400793 31 browser details YourSeq 29 1193 1228 2000 84.4% chr2 + 79062738 79062771 34

Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 http://www.alphaknockout.com/ Gene and protein information: Tnip2 TNFAIP3 interacting protein 2 [ Mus musculus (house mouse) ] Gene ID: 231130, updated on 10-Oct-2019

Gene summary

Official Symbol Tnip2 provided by MGI Official Full Name TNFAIP3 interacting protein 2 provided by MGI Primary source MGI:MGI:2386643 See related Ensembl:ENSMUSG00000059866 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 ABIN-2; AI428870; 1810020H16Rik Expression Ubiquitous expression in adrenal adult (RPKM 22.5), ovary adult (RPKM 19.9) and 28 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 B2 See Tnip2 in Genome Data Viewer Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (34496096..34513979, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (34838745..34856628, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 6 transcripts

Gene: Tnip2 ENSMUSG00000059866

Description TNFAIP3 interacting protein 2 [Source:MGI Symbol;Acc:MGI:2386643] Gene Synonyms 1810020H16Rik, ABIN-2 Location Chromosome 5: 34,496,087-34,513,991 reverse strand. GRCm38:CM000998.2 About this gene This gene has 6 transcripts (splice variants), 161 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tnip2-202 ENSMUST00000087737.9 1987 430aa ENSMUSP00000085030.3 Protein coding CCDS19214 Q99JG7 TSL:1 GENCODE basic APPRIS P1

Tnip2-201 ENSMUST00000030991.13 2022 451aa ENSMUSP00000030991.7 Protein coding - Q80VZ7 TSL:1 GENCODE basic

Tnip2-203 ENSMUST00000114359.1 1642 347aa ENSMUSP00000109999.1 Protein coding - D3Z039 TSL:5 GENCODE basic

Tnip2-204 ENSMUST00000137555.1 900 No protein - Retained intron - - TSL:3

Tnip2-206 ENSMUST00000159102.1 584 No protein - Retained intron - - TSL:2

Tnip2-205 ENSMUST00000143072.1 383 No protein - Retained intron - - TSL:3

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37.91 kb Forward strand 34.49Mb 34.50Mb 34.51Mb 34.52Mb Genes Fam193a-202 >protein coding (Comprehensive set...

Gm43457-201 >TEC

Contigs < AC102620.11 < AC115695.11 Genes (Comprehensive set... < Tnip2-202protein coding

< Tnip2-201protein coding

< Tnip2-203protein coding

< Tnip2-205retained intron

< Tnip2-204retained intron

< Tnip2-206retained intron

Regulatory Build

34.49Mb 34.50Mb 34.51Mb 34.52Mb Reverse strand 37.91 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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

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Transcript: ENSMUST00000030991

< Tnip2-201protein coding

Reverse strand 17.88 kb

ENSMUSP00000030... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam TSG101 and ALIX binding domain of CEP55

NF-kappa-B essential modulator NEMO, CC2-LZ domain PROSITE profiles NEMO, Zinc finger PANTHER PTHR31882

PTHR31882:SF10 Gene3D 1.20.5.990

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

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 451

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

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