https://www.alphaknockout.com Mouse Trim32 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Trim32 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Trim32 gene (NCBI Reference Sequence: NM_053084 ; Ensembl: ENSMUSG00000051675 ) is located on Mouse chromosome 4. 2 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000050850). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Trim32 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-234I22 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 gene trapped allele exhibit mild myopathy with sarcotubular myopathy, decreased fertility, and decreased axon diameter. Mice homozygous for a knock-out allele exhibit impaired adult muscle regeneration and myopathy. Exon 2 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 2. The size of effective cKO region: ~2317 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele T A 5' gRNA region A 3' 1 2 Targeting vector T A A Targeted allele T A A Constitutive KO allele (After Cre recombination) Legends Exon of mouse Trim32 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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(7965bp) | A(25.26% 2012) | C(22.16% 1765) | T(28.71% 2287) | G(23.87% 1901) 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. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr4 + 65610209 65613208 3000 browser details YourSeq 45 2702 2746 3000 100.0% chr3 + 126347505 126347549 45 browser details YourSeq 25 2862 2890 3000 96.3% chr15 + 76113497 76113527 31 browser details YourSeq 21 2587 2608 3000 100.0% chr1 - 10062001 10062023 23 Note: The 3000 bp section upstream of Exon 2 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% chr4 + 65615174 65618173 3000 browser details YourSeq 27 156 199 3000 72.5% chr11 + 80018503 80018538 36 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Trim32 tripartite motif-containing 32 [ Mus musculus (house mouse) ] Gene ID: 69807, updated on 10-Oct-2019 Gene summary Official Symbol Trim32 provided by MGI Official Full Name tripartite motif-containing 32 provided by MGI Primary source MGI:MGI:1917057 See related Ensembl:ENSMUSG00000051675 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 3f3; BBS11; Zfp117; 1810045E12Rik Expression Broad expression in CNS E18 (RPKM 39.0), whole brain E14.5 (RPKM 30.7) and 23 other tissues See more Orthologs human all Genomic context Location: 4 C1; 4 34.43 cM See Trim32 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (65604986..65616240) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (65266020..65277274) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Trim32 ENSMUSG00000051675 Description tripartite motif-containing 32 [Source:MGI Symbol;Acc:MGI:1917057] Gene Synonyms 1810045E12Rik, 3f3, BBS11, Zfp117 Location Chromosome 4: 65,604,986-65,616,238 forward strand. GRCm38:CM000997.2 About this gene This gene has 4 transcripts (splice variants), 183 orthologues, 73 paralogues, is a member of 1 Ensembl protein family and is associated with 20 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Trim32-201 ENSMUST00000050850.13 3194 655aa ENSMUSP00000062277.7 Protein coding CCDS18270 Q3TLR3 Q8CH72 TSL:1 GENCODE basic APPRIS P1 Trim32-202 ENSMUST00000107366.1 3191 655aa ENSMUSP00000102989.1 Protein coding CCDS18270 Q3TLR3 Q8CH72 TSL:1 GENCODE basic APPRIS P1 Trim32-204 ENSMUST00000156922.1 660 183aa ENSMUSP00000121949.1 Protein coding - A2AGS1 CDS 3' incomplete TSL:5 Trim32-203 ENSMUST00000155978.1 516 136aa ENSMUSP00000119579.1 Protein coding - A2AGS0 CDS 3' incomplete TSL:3 31.25 kb Forward strand 65.60Mb 65.61Mb 65.62Mb Genes (Comprehensive set... Trim32-202 >protein coding Trim32-201 >protein coding Trim32-204 >protein coding Trim32-203 >protein coding Contigs AL691456.7 > Genes < Astn2-202protein coding (Comprehensive set... < Astn2-201protein coding Regulatory Build 65.60Mb 65.61Mb 65.62Mb Reverse strand 31.25 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000050850 11.25 kb Forward strand Trim32-201 >protein coding ENSMUSP00000062... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57850 SSF57845 SSF101898 SMART B-box-type zinc finger Zinc finger, RING-type Pfam RING-type zinc-finger, LisH dimerisation motif NHL repeat PROSITE profiles Zinc finger, RING-type NHL repeat, subgroup B-box-type zinc finger PROSITE patterns Zinc finger, RING-type, conserved site PANTHER PTHR25464 PTHR25464:SF3 Gene3D Zinc finger, RING/FYVE/PHD-type Six-bladed beta-propeller, TolB-like CDD cd16587 B-box-type zinc finger cd14961 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 655 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.