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Mouse Cilp Knockout Project (CRISPR/Cas9)

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Mouse Cilp Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cilp Knockout Project (CRISPR/Cas9) Objective: To create a Cilp knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cilp gene (NCBI Reference Sequence: NM_173385 ; Ensembl: ENSMUSG00000042254 ) is located on Mouse chromosome 9. 10 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000048762). Exon 3~9 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 3 starts from the coding region. Exon 3~9 covers 31.63% of the coding region. The size of effective KO region: ~8960 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 6 7 8 9 10 Legends Exon of mouse Cilp Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1191 bp section upstream of Exon 3 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 1809 bp section downstream of Exon 9 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(1191bp) | A(24.43% 291) | C(23.85% 284) | T(27.96% 333) | G(23.76% 283) Note: The 1191 bp section upstream of Exon 3 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(1809bp) | A(29.8% 539) | C(22.17% 401) | T(24.43% 442) | G(23.6% 427) Note: The 1809 bp section downstream of Exon 9 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 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 1191 1 1191 1191 100.0% chr9 + 65265942 65267132 1191 browser details YourSeq 127 849 1055 1191 93.9% chr6 - 84056201 84056486 286 browser details YourSeq 88 966 1056 1191 99.0% chr3 - 89676340 89676432 93 browser details YourSeq 88 932 1055 1191 94.1% chr11 + 98045226 98045507 282 browser details YourSeq 82 966 1055 1191 96.7% chr5 - 71354281 71354383 103 browser details YourSeq 81 919 1033 1191 92.7% chr6 - 113670856 113671418 563 browser details YourSeq 81 972 1177 1191 91.0% chr13 + 43396947 43397162 216 browser details YourSeq 81 964 1051 1191 96.6% chr11 + 106618151 106618240 90 browser details YourSeq 80 935 1049 1191 93.6% chr3 - 152432281 152432461 181 browser details YourSeq 80 939 1032 1191 93.7% chr2 + 165393645 165572293 178649 browser details YourSeq 77 964 1056 1191 91.4% chr2 - 117483075 117483167 93 browser details YourSeq 77 964 1054 1191 93.5% chr12 - 89216122 89216227 106 browser details YourSeq 77 972 1055 1191 96.5% chr11 + 23527888 23527973 86 browser details YourSeq 76 966 1056 1191 92.4% chr5 - 29479982 29480074 93 browser details YourSeq 76 964 1050 1191 94.3% chr4 - 148588557 148588656 100 browser details YourSeq 74 919 1057 1191 92.1% chr7 - 143616528 143616810 283 browser details YourSeq 72 966 1061 1191 91.8% chr5 - 31123876 31123973 98 browser details YourSeq 69 976 1055 1191 93.8% chr11 - 114836303 114836388 86 browser details YourSeq 69 820 1039 1191 92.7% chr13 + 46416951 46417378 428 browser details YourSeq 68 804 992 1191 86.1% chr16 - 10395842 10396456 615 Note: The 1191 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 1809 1 1809 1809 100.0% chr9 + 65276003 65277811 1809 browser details YourSeq 116 972 1740 1809 92.0% chr10 - 117443272 117449498 6227 browser details YourSeq 91 782 1011 1809 83.9% chr17 + 42708139 42708324 186 browser details YourSeq 87 1399 1522 1809 94.8% chr1 - 80482286 80482825 540 browser details YourSeq 80 787 1006 1809 80.4% chr7 - 116711218 116711417 200 browser details YourSeq 79 780 1016 1809 77.8% chr3 - 88559204 88559394 191 browser details YourSeq 78 779 1006 1809 74.8% chr1 - 55180923 55181127 205 browser details YourSeq 74 158 682 1809 73.0% chr19 + 9990750 9990851 102 browser details YourSeq 72 792 1005 1809 76.7% chr13 - 38175366 38175566 201 browser details YourSeq 70 1456 1557 1809 90.7% chr12 + 80732916 80733057 142 browser details YourSeq 69 854 990 1809 74.8% chr3 - 138155898 138156016 119 browser details YourSeq 69 652 892 1809 94.9% chr14 + 63257402 63257787 386 browser details YourSeq 68 787 1005 1809 88.7% chr16 + 64988630 64988849 220 browser details YourSeq 67 923 1005 1809 88.9% chrX + 139689567 139689648 82 browser details YourSeq 65 785 1005 1809 82.9% chr1 - 192738905 193139476 400572 browser details YourSeq 63 881 1007 1809 78.0% chr11 + 106197767 106197870 104 browser details YourSeq 62 922 1002 1809 89.8% chr2 - 181376580 181376668 89 browser details YourSeq 62 794 959 1809 80.8% chr2 - 156451126 156451281 156 browser details YourSeq 62 826 1016 1809 90.8% chr6 + 120241987 120242375 389 browser details YourSeq 62 823 950 1809 83.4% chr5 + 6062795 6062913 119 Note: The 1809 bp section downstream of Exon 9 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Cilp cartilage intermediate layer protein, nucleotide pyrophosphohydrolase [ Mus musculus (house mouse) ] Gene ID: 214425, updated on 12-Aug-2019 Gene summary Official Symbol Cilp provided by MGI Official Full Name cartilage intermediate layer protein, nucleotide pyrophosphohydrolase provided by MGI Primary source MGI:MGI:2444507 See related Ensembl:ENSMUSG00000042254 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 CILP-1; 9830114D16; C130036G17Rik Expression Broad expression in genital fat pad adult (RPKM 10.5), subcutaneous fat pad adult (RPKM 8.2) and 16 other tissues See Orthologs more human all Genomic context Location: 9; 9 C See Cilp in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (65265180..65280614) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (65112987..65128412) Chromosome 9 - NC_000075.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Cilp ENSMUSG00000042254 Description cartilage intermediate layer protein, nucleotide pyrophosphohydrolase [Source:MGI Symbol;Acc:MGI:2444507] Gene Synonyms C130036G17Rik Location Chromosome 9: 65,265,180-65,280,605 forward strand. GRCm38:CM001002.2 About this gene This gene has 2 transcripts (splice variants), 194 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cilp- ENSMUST00000048762.7 4153 1250aa ENSMUSP00000036631.1 Protein coding CCDS23287 A0A0R4J0F8 TSL:1 201 GENCODE basic APPRIS P1 Cilp- ENSMUST00000141382.1 4128 74aa ENSMUSP00000121326.1 Nonsense mediated - D6RD06 TSL:5 202 decay 35.43 kb Forward strand 65.26Mb 65.27Mb 65.28Mb 65.29Mb Genes (Comprehensive set... Cilp-201 >protein coding Cilp-202 >nonsense mediated decay Contigs AC110235.13 > Regulatory Build 65.26Mb 65.27Mb 65.28Mb 65.29Mb Reverse strand 35.43 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000048762 15.43 kb Forward strand Cilp-201 >protein coding ENSMUSP00000036... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily Thrombospondin type-1 (TSP1) repeat superfamily SMART Immunoglobulin subtype Thrombospondin type-1 (TSP1) repeat Immunoglobulin subtype 2 Pfam WxxW domain PF13927 Thrombospondin type-1 (TSP1) repeat PROSITE profiles Immunoglobulin-like domain Thrombospondin type-1 (TSP1) repeat PANTHER Cartilage intermediate layer protein 1/2 PTHR15031:SF3 Gene3D Immunoglobulin-like fold Thrombospondin type-1 (TSP1) repeat superfamily CDD cd00096 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained frameshift variant missense variant synonymous variant Scale bar 0 200 400 600 800 1000 1250 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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