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Mouse Tifa Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Tifa gene (NCBI Reference Sequence: NM_145133.3 ; Ensembl: ENSMUSG00000046688 ) is located on Mouse chromosome 3. 3 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000054483). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tifa gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-321J10 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 3 covers 100.0% of the coding region. Start codon is in exon 3, and stop codon is in exon 3. The size of intron 2 for 5'-loxP site insertion: 5039 bp. The size of effective cKO region: ~2558 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 3 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tifa 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(7052bp) | A(28.72% 2025) | C(20.76% 1464) | T(29.52% 2082) | G(21.0% 1481)

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% chr3 + 127793333 127796332 3000 browser details YourSeq 192 10 670 3000 85.8% chr2 + 156171748 156172210 463 browser details YourSeq 184 12 659 3000 84.7% chr14 - 21699334 21699853 520 browser details YourSeq 151 90 740 3000 86.4% chr2 - 126689674 126690234 561 browser details YourSeq 147 572 755 3000 93.0% chr1 - 34372417 34372600 184 browser details YourSeq 146 589 755 3000 96.2% chr11 - 77639066 77639422 357 browser details YourSeq 144 10 171 3000 94.5% chr13 - 78472243 78472404 162 browser details YourSeq 144 589 755 3000 90.0% chr4 + 124475981 124476139 159 browser details YourSeq 142 4 170 3000 92.9% chr5 - 135647765 135647932 168 browser details YourSeq 142 10 169 3000 94.4% chr5 - 21035116 21035275 160 browser details YourSeq 142 588 755 3000 90.2% chr19 + 9887609 9887771 163 browser details YourSeq 141 1 169 3000 92.3% chr6 - 37813275 37813445 171 browser details YourSeq 141 589 755 3000 90.7% chr19 - 44679224 44679386 163 browser details YourSeq 141 589 763 3000 87.8% chr12 - 78282568 78282731 164 browser details YourSeq 140 10 171 3000 94.4% chr9 + 110804061 110804223 163 browser details YourSeq 138 590 760 3000 89.9% chrX - 20970668 20970831 164 browser details YourSeq 138 10 175 3000 94.3% chr3 - 135519303 135519480 178 browser details YourSeq 137 10 170 3000 92.6% chr7 - 38253104 38253264 161 browser details YourSeq 137 589 754 3000 89.0% chr18 - 15171760 15171922 163 browser details YourSeq 137 10 170 3000 92.6% chr3 + 32531702 32531862 161

Note: The 3000 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 3000 1 3000 3000 100.0% chr3 + 127797135 127800134 3000 browser details YourSeq 240 1450 1824 3000 87.7% chr6 - 14292272 14292641 370 browser details YourSeq 221 1442 1824 3000 84.0% chr6 + 50486751 50487086 336 browser details YourSeq 217 1475 1821 3000 87.1% chr7 - 31027369 31027676 308 browser details YourSeq 213 1495 1885 3000 81.6% chr9 - 50360881 50361266 386 browser details YourSeq 210 1497 1815 3000 86.7% chr10 + 122818492 122818799 308 browser details YourSeq 208 1436 1821 3000 85.6% chr16 - 31350367 31350718 352 browser details YourSeq 204 1495 1800 3000 86.3% chr4 - 101307509 101307806 298 browser details YourSeq 197 1499 1824 3000 85.2% chr13 + 59438999 59439283 285 browser details YourSeq 196 1474 1821 3000 82.4% chr1 - 181501193 181501496 304 browser details YourSeq 194 1443 1763 3000 86.1% chr17 + 79819966 79820245 280 browser details YourSeq 183 1456 1768 3000 84.3% chr3 - 103187627 103187926 300 browser details YourSeq 182 1475 1824 3000 85.4% chr18 - 62794061 62794367 307 browser details YourSeq 182 1496 1800 3000 83.8% chr18 + 7545712 7545970 259 browser details YourSeq 181 1442 1764 3000 89.9% chr5 + 102129308 102129635 328 browser details YourSeq 180 1558 1800 3000 87.5% chr8 - 118030818 118031058 241 browser details YourSeq 179 1471 1824 3000 86.8% chr4 - 49500066 49500381 316 browser details YourSeq 177 1494 1824 3000 85.2% chr8 + 55618861 55619205 345 browser details YourSeq 177 1496 1784 3000 85.6% chr6 + 106870025 106870303 279 browser details YourSeq 175 1494 1800 3000 87.2% chr3 + 133030467 133030770 304

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: Tifa TRAF-interacting protein with forkhead-associated domain [ Mus musculus (house mouse) ] Gene ID: 211550, updated on 26-Jun-2020

Gene summary

Official Symbol Tifa provided by MGI Official Full Name TRAF-interacting protein with forkhead-associated domain provided by MGI Primary source MGI:MGI:2182965 See related Ensembl:ENSMUSG00000046688 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 T2bp Expression Biased expression in large intestine adult (RPKM 20.9), colon adult (RPKM 4.1) and 12 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 G2 See Tifa in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (127788875..127798394)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (127492831..127501307)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 5 transcripts

Gene: Tifa ENSMUSG00000046688

Description TRAF-interacting protein with forkhead-associated domain [Source:MGI Symbol;Acc:MGI:2182965] Gene Synonyms T2bp Location Chromosome 3: 127,789,805-127,832,164 forward strand. GRCm38:CM000996.2 About this gene This gene has 5 transcripts (splice variants), 272 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tifa- ENSMUST00000171621.2 2547 184aa ENSMUSP00000127700.1 Protein coding CCDS38628 I3PQW8 TSL:1 205 Q793I8 GENCODE basic APPRIS P1

Tifa- ENSMUST00000054483.13 2245 184aa ENSMUSP00000054036.7 Protein coding CCDS38628 I3PQW8 TSL:1 201 Q793I8 GENCODE basic APPRIS P1

Tifa- ENSMUST00000163775.5 1646 184aa ENSMUSP00000132309.1 Protein coding CCDS38628 I3PQW8 TSL:1 202 Q793I8 GENCODE basic APPRIS P1

Tifa- ENSMUST00000164447.2 1538 184aa ENSMUSP00000126692.1 Protein coding CCDS38628 I3PQW8 TSL:1 203 Q793I8 GENCODE basic APPRIS P1

Tifa- ENSMUST00000166778.2 452 No - Processed - - TSL:5 204 protein transcript

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62.36 kb Forward strand 127.78Mb 127.80Mb 127.82Mb 127.84Mb Genes (Comprehensive set... Tifa-201 >protein coding Gm43653-201 >antisense

Tifa-204 >processed transcript

Tifa-202 >protein coding

Tifa-203 >protein coding

Tifa-205 >protein coding

Contigs < AC163391.4 Genes < Alpk1-201protein coding < Ap1ar-210protein coding (Comprehensive set...

< Alpk1-203retained intron < Ap1ar-201protein coding

< Alpk1-204retained intron < Ap1ar-207retained intron < Ap1ar-203retained intron < Gm43654-201TEC

< Alpk1-205processed transcript < Ap1ar-204retained intro

< Ap1ar-208retained intron

< Ap1ar-206retained intron

< Ap1ar-209retained intron

< Ap1ar-205retained intron

Regulatory Build

127.78Mb 127.80Mb 127.82Mb 127.84Mb Reverse strand 62.36 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000054483

8.59 kb Forward strand

Tifa-201 >protein coding

ENSMUSP00000054... Superfamily SMAD/FHA domain superfamily Pfam Forkhead-associated (FHA) domain PROSITE profiles Forkhead-associated (FHA) domain PANTHER TRAF-interacting protein with FHA domain-containing protein

PTHR31266:SF2 Gene3D 2.60.200.20 CDD Forkhead-associated (FHA) domain

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 184

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

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