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Mouse Relt Knockout Project (CRISPR/Cas9)

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Mouse Relt Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Relt Knockout Project (CRISPR/Cas9) Objective: To create a Relt knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Relt gene (NCBI Reference Sequence: NM_177073 ; Ensembl: ENSMUSG00000008318 ) is located on Mouse chromosome 7. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000008462). Exon 2~11 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 with a premature termination codon resulting in a null allele exhibit incisor and molar enamel malformations. Exon 2 starts from about 0.08% of the coding region. Exon 2~11 covers 100.0% of the coding region. The size of effective KO region: ~5877 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 9 10 11 Legends Exon of mouse Relt 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. 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 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(20.25% 405) | C(24.15% 483) | T(25.7% 514) | G(29.9% 598) 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(23.4% 468) | C(24.05% 481) | T(24.75% 495) | G(27.8% 556) 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 - 100853112 100855111 2000 browser details YourSeq 24 132 161 2000 88.5% chr2 - 33371714 33371742 29 browser details YourSeq 20 1602 1623 2000 95.5% chr1 - 62352301 62352322 22 browser details YourSeq 20 1260 1279 2000 100.0% chr1 + 37664909 37664928 20 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 - 100845233 100847232 2000 browser details YourSeq 1186 113 1386 2000 96.8% chr7 - 25420589 25421867 1279 browser details YourSeq 938 1 1383 2000 87.3% chrX + 154436752 154450469 13718 browser details YourSeq 35 1638 1706 2000 87.5% chr2 + 43178225 43178291 67 browser details YourSeq 23 894 916 2000 100.0% chr10 + 59660249 59660271 23 browser details YourSeq 22 1913 1934 2000 100.0% chr8 - 90563831 90563852 22 browser details YourSeq 22 781 802 2000 100.0% chr1 + 94177961 94177982 22 browser details YourSeq 21 1583 1603 2000 100.0% chr8 + 84825821 84825841 21 browser details YourSeq 21 273 293 2000 100.0% chr17 + 19124906 19124926 21 browser details YourSeq 21 1108 1128 2000 100.0% chr15 + 72355856 72355876 21 browser details YourSeq 21 1016 1036 2000 100.0% chr14 + 75615736 75615756 21 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: Relt RELT tumor necrosis factor receptor [ Mus musculus (house mouse) ] Gene ID: 320100, updated on 10-Oct-2019 Gene summary Official Symbol Relt provided by MGI Official Full Name RELT tumor necrosis factor receptor provided by MGI Primary source MGI:MGI:2443373 See related Ensembl:ENSMUSG00000008318 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 Tnfrsf19l; E430021K24Rik Expression Broad expression in spleen adult (RPKM 9.5), thymus adult (RPKM 6.5) and 24 other tissues See more Orthologs human all Genomic context Location: 7; 7 E2 See Relt in Genome Data Viewer Exon count: 17 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (100845847..100863489, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (107994362..108011927, complement) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Relt ENSMUSG00000008318 Description RELT tumor necrosis factor receptor [Source:MGI Symbol;Acc:MGI:2443373] Gene Synonyms E430021K24Rik, Tnfrsf19l Location Chromosome 7: 100,845,847-100,863,446 reverse strand. GRCm38:CM001000.2 About this gene This gene has 6 transcripts (splice variants), 177 orthologues, 21 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Relt- ENSMUST00000008462.10 2851 436aa ENSMUSP00000008462.3 Protein coding CCDS21506 Q8BX43 TSL:1 201 GENCODE basic APPRIS P1 Relt- ENSMUST00000136231.1 516 119aa ENSMUSP00000121443.1 Protein coding - D3Z306 CDS 3' incomplete 202 TSL:2 Relt- ENSMUST00000139604.7 459 123aa ENSMUSP00000119208.1 Protein coding - D3YWL9 CDS 3' incomplete 203 TSL:5 Relt- ENSMUST00000156855.7 428 123aa ENSMUSP00000120042.1 Protein coding - D3YWL9 CDS 3' incomplete 206 TSL:5 Relt- ENSMUST00000142885.1 346 58aa ENSMUSP00000121049.1 Protein coding - D3YTN8 CDS 3' incomplete 204 TSL:3 Relt- ENSMUST00000155413.8 1966 119aa ENSMUSP00000118150.2 Nonsense mediated decay - D6RIM1 TSL:1 205 Page 7 of 9 https://www.alphaknockout.com 37.60 kb Forward strand 100.84Mb 100.85Mb 100.86Mb 100.87Mb Genes Fam168a-202 >protein coding (Comprehensive set... Fam168a-204 >protein coding Fam168a-201 >protein coding Fam168a-205 >protein coding Fam168a-206 >protein coding Fam168a-210 >protein coding Contigs AC150744.2 > Genes (Comprehensive set... < Relt-201protein coding < Arhgef17-201protein coding < Relt-205nonsense mediated decay < Arhgef17-204protein coding < Relt-203protein coding < Relt-206protein coding < Relt-202protein coding < Relt-204protein coding Regulatory Build 100.84Mb 100.85Mb 100.86Mb 100.87Mb Reverse strand 37.60 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000008462 < Relt-201protein coding Reverse strand 17.60 kb ENSMUSP00000008... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... SMART TNFR/NGFR cysteine-rich region Prints Tumour necrosis factor receptor 19-like Pfam TNFR/NGFR cysteine-rich region TNF receptor family, RELT PANTHER Tumour necrosis factor receptor 19-like Gene3D 2.10.50.10 CDD Tumor necrosis factor receptor 19-like, N-terminal All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 436 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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