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Mouse Trim6 Knockout Project (CRISPR/Cas9)

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Mouse Trim6 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Trim6 Knockout Project (CRISPR/Cas9) Objective: To create a Trim6 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Trim6 gene (NCBI Reference Sequence: NM_001013616 ; Ensembl: ENSMUSG00000072244 ) is located on Mouse chromosome 7. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000098180). Exon 2~8 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: Exon 2 starts from about 0.07% of the coding region. Exon 2~8 covers 100.0% of the coding region. The size of effective KO region: ~7372 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 Legends Exon of mouse Trim6 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of stop codon 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.3% 526) | C(20.45% 409) | T(30.2% 604) | G(23.05% 461) Note: The 2000 bp section upstream of start codon 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(26.3% 526) | C(19.0% 380) | T(35.15% 703) | G(19.55% 391) Note: The 2000 bp section downstream of stop codon 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 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr7 + 104223559 104225558 2000 browser details YourSeq 774 104 907 2000 98.3% chr7 + 104324884 104325941 1058 browser details YourSeq 87 650 978 2000 76.7% chr3 + 127587270 127587430 161 browser details YourSeq 66 875 972 2000 87.5% chr1 + 133836768 133836880 113 browser details YourSeq 64 854 983 2000 86.4% chr7 + 75058870 75059001 132 browser details YourSeq 52 817 974 2000 80.4% chr16 - 29161400 29161546 147 browser details YourSeq 52 1174 1247 2000 90.7% chr9 + 95690514 95690872 359 browser details YourSeq 50 812 888 2000 80.0% chrX - 98841345 98841417 73 browser details YourSeq 49 854 996 2000 84.6% chr19 - 24026829 24026972 144 browser details YourSeq 49 859 970 2000 88.2% chr11 + 70430400 70430510 111 browser details YourSeq 46 817 918 2000 82.8% chr10 - 121321230 121321328 99 browser details YourSeq 45 883 971 2000 75.3% chr1 - 88884642 88884730 89 browser details YourSeq 45 854 976 2000 94.2% chr1 + 82215789 82215912 124 browser details YourSeq 44 1186 1257 2000 73.6% chr11 - 78340566 78340618 53 browser details YourSeq 44 833 959 2000 90.6% chr11 + 116953726 116953854 129 browser details YourSeq 43 924 972 2000 93.9% chr12 - 20016863 20016911 49 browser details YourSeq 43 933 980 2000 95.9% chr12 + 80488390 80488438 49 browser details YourSeq 43 924 972 2000 93.9% chr12 + 23240071 23240119 49 browser details YourSeq 42 1207 1274 2000 83.1% chr10 - 45773994 45774066 73 browser details YourSeq 42 937 981 2000 97.8% chr9 + 67686034 67686079 46 Note: The 2000 bp section upstream of start codon 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% chr7 + 104232931 104234930 2000 browser details YourSeq 72 170 279 2000 93.0% chr12 - 72994843 72994965 123 browser details YourSeq 60 181 346 2000 91.7% chr6 - 39470779 39471107 329 browser details YourSeq 60 210 360 2000 91.9% chr10 + 111970516 111970765 250 browser details YourSeq 51 130 331 2000 96.5% chr14 - 51517431 51517783 353 browser details YourSeq 48 151 213 2000 89.9% chr10 + 99696608 99696678 71 browser details YourSeq 42 198 279 2000 93.9% chr17 + 16429732 16429824 93 browser details YourSeq 39 291 371 2000 90.0% chr5 - 121718138 121718241 104 browser details YourSeq 36 302 349 2000 83.4% chr2 - 69362704 69362749 46 browser details YourSeq 33 303 355 2000 86.5% chr11 + 79171821 79171871 51 browser details YourSeq 33 210 264 2000 92.4% chr11 + 57666586 57666640 55 browser details YourSeq 31 302 337 2000 97.2% chr12 - 35198481 35198524 44 browser details YourSeq 30 456 497 2000 85.8% chr18 + 24008380 24008421 42 browser details YourSeq 30 1506 1537 2000 100.0% chr10 + 44362002 44362039 38 browser details YourSeq 29 312 342 2000 90.0% chr17 + 13524536 13524565 30 browser details YourSeq 28 217 250 2000 91.2% chr13 + 92517131 92517164 34 browser details YourSeq 24 411 440 2000 90.0% chr19 + 50119046 50119075 30 browser details YourSeq 22 309 330 2000 100.0% chr14 + 80503602 80503623 22 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Trim6 tripartite motif-containing 6 [ Mus musculus (house mouse) ] Gene ID: 94088, updated on 12-Aug-2019 Gene summary Official Symbol Trim6 provided by MGI Official Full Name tripartite motif-containing 6 provided by MGI Primary source MGI:MGI:2137352 See related Ensembl:ENSMUSG00000072244 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 D7Ertd684e; C430046K18Rik Expression Biased expression in placenta adult (RPKM 14.8) and colon adult (RPKM 1.4) See more Genomic context Location: 7 E3; 7 55.55 cM See Trim6 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (104218795..104235152) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (111367309..111383666) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Trim6 ENSMUSG00000072244 Description tripartite motif-containing 6 [Source:MGI Symbol;Acc:MGI:2137352] Gene Synonyms C430046K18Rik, D7Ertd684e Location Chromosome 7: 104,218,793-104,235,152 forward strand. GRCm38:CM001000.2 About this gene This gene has 6 transcripts (splice variants), 75 orthologues, 73 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 Trim6-201 ENSMUST00000098180.9 3827 488aa ENSMUSP00000095782.3 Protein coding CCDS40065 Q8BGE7 TSL:1 GENCODE basic APPRIS P1 Trim6-206 ENSMUST00000214578.1 1506 462aa ENSMUSP00000149805.1 Protein coding - A0A1L1SS93 TSL:5 GENCODE basic Trim6-205 ENSMUST00000153324.1 622 148aa ENSMUSP00000119932.1 Protein coding - D3Z7U8 CDS 3' incomplete TSL:3 Trim6-202 ENSMUST00000128493.1 536 125aa ENSMUSP00000118859.1 Protein coding - D3YY77 CDS 3' incomplete TSL:3 Trim6-203 ENSMUST00000144455.1 520 113aa ENSMUSP00000116233.1 Protein coding - D3Z676 CDS 3' incomplete TSL:3 Trim6-204 ENSMUST00000144538.1 1490 No protein - Retained intron - - TSL:1 Page 7 of 9 https://www.alphaknockout.com 36.36 kb Forward strand 104.21Mb 104.22Mb 104.23Mb 104.24Mb Genes (Comprehensive set... Trim6-204 >retained intron Trim34a-203 >protein coding Trim6-201 >protein coding Trim34a-201 >protein coding Trim6-203 >protein coding Trim34a-202 >protein coding Trim6-202 >protein coding Trim6-205 >protein coding Trim6-206 >protein coding Contigs < AC123830.5 Genes < Gm8667-201processed pseudogene (Comprehensive set... Regulatory Build 104.21Mb 104.22Mb 104.23Mb 104.24Mb Reverse strand 36.36 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript pseudogene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000098180 16.36 kb Forward strand Trim6-201 >protein coding ENSMUSP00000095... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57850 SSF57845 Concanavalin A-like lectin/glucanase domain superfamily SMART Zinc finger, RING-type SPRY domain B-box-type zinc finger SPRY-associated Prints Butyrophylin-like, SPRY domain Pfam RING-type zinc-finger, LisH dimerisation motif SPRY domain B-box-type zinc finger PROSITE profiles B-box-type zinc finger B30.2/SPRY domain Zinc finger, RING-type PROSITE patterns Zinc finger, RING-type, conserved site PANTHER PTHR24103:SF51 PTHR24103 Gene3D 3.30.40.200 2.60.120.920 Zinc finger, RING/FYVE/PHD-type CDD cd16591 B-box-type zinc finger TRIM6, PRY/SPRY domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 488 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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