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Mouse Trim31 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Trim31 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Trim31 gene (NCBI Reference Sequence: NM_146077 ; Ensembl: ENSMUSG00000058063 ) is located on Mouse chromosome 17. 8 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000078438). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Trim31 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-383N9 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: Homozygous knockout exacerbates the immune response after induced inflammation and in induced peritonitis and reduces the immune response in induced colitis.

Exon 4 starts from about 48.78% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 3534 bp, and the size of intron 5 for 3'-loxP site insertion: 1237 bp. The size of effective cKO region: ~1109 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 4 5 6 7 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Trim31 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(7609bp) | A(27.51% 2093) | C(22.75% 1731) | T(28.47% 2166) | G(21.28% 1619)

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% chr17 + 36902165 36905164 3000 browser details YourSeq 240 1006 1329 3000 87.8% chr5 - 119888199 119912240 24042 browser details YourSeq 231 1012 1329 3000 88.2% chr9 + 41033002 41033318 317 browser details YourSeq 223 549 1323 3000 84.4% chrX - 94515192 94515953 762 browser details YourSeq 218 1028 1329 3000 86.9% chr11 - 113321071 113321374 304 browser details YourSeq 211 1047 1329 3000 87.6% chr14 + 48532364 48921261 388898 browser details YourSeq 211 1012 1329 3000 84.2% chr10 + 59922420 59922738 319 browser details YourSeq 210 1012 1329 3000 87.0% chr1 + 157091529 157091848 320 browser details YourSeq 207 1063 1505 3000 85.0% chr11 - 8749830 8750231 402 browser details YourSeq 207 1051 1335 3000 86.7% chr7 + 40370714 40371006 293 browser details YourSeq 206 1051 1329 3000 87.8% chr2 - 169201441 169201719 279 browser details YourSeq 205 552 1329 3000 82.4% chr5 + 138866601 138867297 697 browser details YourSeq 202 1047 1329 3000 87.7% chr5 - 146889949 146890232 284 browser details YourSeq 201 1030 1328 3000 84.5% chrX - 153806536 153806834 299 browser details YourSeq 201 1000 1327 3000 85.1% chr7 + 142078682 142079012 331 browser details YourSeq 201 1005 1310 3000 86.0% chr14 + 59812475 59812780 306 browser details YourSeq 200 1047 1329 3000 86.2% chr13 - 57992085 57992367 283 browser details YourSeq 199 1025 1329 3000 86.5% chr3 - 44296922 44297245 324 browser details YourSeq 196 1051 1328 3000 87.7% chr9 - 45214721 45214999 279 browser details YourSeq 196 1047 1329 3000 86.3% chr5 + 32511272 32511555 284

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% chr17 + 36906274 36909273 3000 browser details YourSeq 165 2276 2796 3000 86.5% chr16 - 92758210 92758599 390 browser details YourSeq 153 2277 2796 3000 85.0% chr12 - 101777209 101777462 254 browser details YourSeq 127 2276 2794 3000 82.1% chr1 - 139345435 139345750 316 browser details YourSeq 123 2277 2630 3000 95.0% chr15 + 37763941 37764349 409 browser details YourSeq 121 2524 2796 3000 87.4% chr16 - 92758187 92758432 246 browser details YourSeq 118 2552 2926 3000 83.5% chrX - 51557887 51558155 269 browser details YourSeq 111 2311 2789 3000 78.4% chr10 + 123347335 123347724 390 browser details YourSeq 103 2377 2774 3000 91.9% chr10 - 119009793 119013320 3528 browser details YourSeq 98 2281 2758 3000 83.7% chr4 - 119047475 119047884 410 browser details YourSeq 97 2309 2794 3000 77.5% chr6 + 30339369 30339509 141 browser details YourSeq 96 2377 2796 3000 79.1% chr8 - 53596578 53596785 208 browser details YourSeq 96 2528 2796 3000 94.4% chr18 - 73887980 73888363 384 browser details YourSeq 95 2529 2795 3000 88.8% chr6 - 121286129 121286389 261 browser details YourSeq 93 2313 2789 3000 77.1% chr1 - 130563687 130563877 191 browser details YourSeq 91 2277 2796 3000 78.2% chr10 + 75112125 75112229 105 browser details YourSeq 88 2526 2797 3000 95.0% chr8 - 14604734 14605053 320 browser details YourSeq 86 2468 2793 3000 77.4% chr18 - 76742111 76742340 230 browser details YourSeq 85 2528 2796 3000 91.0% chr10 - 77328251 77328760 510 browser details YourSeq 84 2526 2690 3000 92.8% chr18 - 73888052 73888335 284

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Trim31 tripartite motif-containing 31 [ Mus musculus (house mouse) ] Gene ID: 224762, updated on 10-Oct-2019

Gene summary

Official Symbol Trim31 provided by MGI Official Full Name tripartite motif-containing 31 provided by MGI Primary source MGI:MGI:2385051 See related Ensembl:ENSMUSG00000058063 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 RNF; HCG1; HCGI; BC026666 Expression Biased expression in large intestine adult (RPKM 25.8), small intestine adult (RPKM 19.0) and 2 other tissuesS ee more Orthologs human all

Genomic context

Location: 17; 17 B1 See Trim31 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (36898130..36910217)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (37035075..37047162)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 1 transcript

Gene: Trim31 ENSMUSG00000058063

Description tripartite motif-containing 31 [Source:MGI Symbol;Acc:MGI:2385051] Gene Synonyms HCG1 Location Chromosome 17: 36,898,118-36,910,214 forward strand. GRCm38:CM001010.2 About this gene This gene has 1 transcript (splice variant), 54 orthologues, 73 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Trim31-201 ENSMUST00000078438.4 2257 507aa ENSMUSP00000077535.4 Protein coding CCDS28728 Q8R0K2 TSL:1 GENCODE basic APPRIS P1

32.10 kb Forward strand 36.89Mb 36.90Mb 36.91Mb 36.92Mb Genes Trim31-201 >protein coding (Comprehensive set...

Contigs < CR956641.9 Genes < Trim40-201protein coding (Comprehensive set...

< Trim40-203protein coding

< Trim40-204retained intron

< Trim40-202lncRNA

Regulatory Build

36.89Mb 36.90Mb 36.91Mb 36.92Mb Reverse strand 32.10 kb

Regulation Legend CTCF Open Chromatin Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000078438

12.10 kb Forward strand

Trim31-201 >protein coding

ENSMUSP00000077... Low complexity (Seg) Superfamily SSF57850 SSF57845 Concanavalin A-like lectin/glucanase domain superfamily

SMART B-box-type zinc finger SPRY domain

Zinc finger, RING-type Prints Butyrophylin-like, SPRY domain Pfam PF15227 B-box-type zinc finger SPRY domain

PROSITE profiles Zinc finger, RING-type B30.2/SPRY domain

B-box-type zinc finger PROSITE patterns Zinc finger, RING-type, conserved site PANTHER PTHR24103

PTHR24103:SF87 Gene3D Zinc finger, RING/FYVE/PHD-type 2.60.120.920

3.30.40.200 CDD B-box-type zinc finger

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 507

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

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