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Mouse Ly6g6e Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ly6g6e Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ly6g6e Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ly6g6e conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ly6g6e gene (NCBI Reference Sequence: NM_027366 ; Ensembl: ENSMUSG00000013766 ) is located on Mouse chromosome 17. 3 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 3 (Transcript: ENSMUST00000013910). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ly6g6e gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-349B4 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: Exon 2~3 covers 87.06% of the coding region. Start codon is in exon 1, and stop codon is in exon 3. The size of intron 1 for 5'-loxP site insertion: 630 bp. The size of effective cKO region: ~1718 bp. The cKO 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 2 3 6 5 4 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Ly6g6e cKO region Exon of mouse Ly6g6f loxP site Page 2 of 8 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(6946bp) | A(23.5% 1632) | C(26.26% 1824) | T(25.25% 1754) | G(24.99% 1736) 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 8 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% chr17 + 35074588 35077587 3000 browser details YourSeq 138 726 1379 3000 90.2% chr11 + 94176246 94437152 260907 browser details YourSeq 124 753 1378 3000 84.5% chr17 - 46189483 46567027 377545 browser details YourSeq 84 527 1031 3000 84.5% chr16 + 9749483 9749995 513 browser details YourSeq 78 791 1010 3000 81.3% chr15 + 73812788 73812988 201 browser details YourSeq 76 921 1036 3000 80.9% chr2 + 77301060 77301174 115 browser details YourSeq 67 1257 1379 3000 94.8% chrX - 153729355 153729549 195 browser details YourSeq 64 938 1037 3000 82.0% chr11 - 32742036 32742135 100 browser details YourSeq 63 938 1032 3000 83.2% chr12 - 85989404 85989498 95 browser details YourSeq 60 921 1006 3000 82.4% chr16 + 94616456 94616540 85 browser details YourSeq 60 795 1025 3000 73.9% chr14 + 54307927 54308133 207 browser details YourSeq 59 922 1027 3000 81.7% chr7 + 111542040 111542144 105 browser details YourSeq 58 1330 1796 3000 72.5% chr9 + 27274151 27274550 400 browser details YourSeq 58 824 1028 3000 79.2% chr6 + 94709665 94709846 182 browser details YourSeq 57 1323 1391 3000 91.4% chr5 - 138765403 138765471 69 browser details YourSeq 56 921 1029 3000 77.4% chr18 - 5270834 5270941 108 browser details YourSeq 56 932 1015 3000 80.8% chr17 - 79934759 79934841 83 browser details YourSeq 56 629 974 3000 75.8% chr14 - 24529010 24529311 302 browser details YourSeq 55 638 1251 3000 64.6% chr6 - 49760181 49760290 110 browser details YourSeq 55 1323 1386 3000 93.8% chr15 - 102455272 102455480 209 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% chr17 + 35078284 35081283 3000 browser details YourSeq 83 713 898 3000 83.5% chr7 - 142245811 142245998 188 browser details YourSeq 81 739 1117 3000 90.8% chr19 + 7252088 7253101 1014 browser details YourSeq 73 1117 1196 3000 96.3% chr1 - 82625809 82625892 84 browser details YourSeq 69 1116 1193 3000 96.2% chr2 - 169401031 169401444 414 browser details YourSeq 68 710 1127 3000 72.2% chr11 - 33058313 33058476 164 browser details YourSeq 64 701 1160 3000 70.3% chr13 + 48387373 48387521 149 browser details YourSeq 62 713 875 3000 93.2% chr4 - 133511958 133512326 369 browser details YourSeq 60 1099 1170 3000 93.1% chr10 + 69679486 69679562 77 browser details YourSeq 59 706 895 3000 83.6% chr2 + 156326557 156326737 181 browser details YourSeq 57 710 1114 3000 89.1% chr4 - 104400035 104400458 424 browser details YourSeq 57 701 774 3000 90.3% chr10 - 59833467 59833542 76 browser details YourSeq 56 710 897 3000 88.0% chr14 + 122185954 122186148 195 browser details YourSeq 53 743 833 3000 93.5% chr11 - 116101489 116101582 94 browser details YourSeq 53 1095 1166 3000 88.8% chr1 - 150610044 150610137 94 browser details YourSeq 51 790 887 3000 90.0% chr15 - 81289696 81289792 97 browser details YourSeq 50 710 898 3000 70.8% chr10 - 75901981 75902131 151 browser details YourSeq 50 710 792 3000 93.2% chr7 + 30800751 30800837 87 browser details YourSeq 50 701 885 3000 75.0% chr13 + 64324885 64325038 154 browser details YourSeq 49 705 766 3000 89.3% chr1 - 181565242 181565302 61 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Ly6g6e lymphocyte antigen 6 complex, locus G6E [ Mus musculus (house mouse) ] Gene ID: 70274, updated on 12-Aug-2019 Gene summary Official Symbol Ly6g6e provided by MGI Official Full Name lymphocyte antigen 6 complex, locus G6E provided by MGI Primary source MGI:MGI:1917524 See related Ensembl:ENSMUSG00000013766 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 G6e; 2310011I02Rik Expression Biased expression in stomach adult (RPKM 36.3), colon adult (RPKM 18.8) and 8 other tissues See more Genomic context Location: 17; 17 B1 See Ly6g6e in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (35076868..35078804) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (35213887..35215749) Chromosome 17 - NC_000083.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Ly6g6e ENSMUSG00000013766 Description lymphocyte antigen 6 complex, locus G6E [Source:MGI Symbol;Acc:MGI:1917524] Gene Synonyms 2310011I02Rik, G6e Location Chromosome 17: 35,076,902-35,078,804 forward strand. GRCm38:CM001010.2 About this gene This gene has 4 transcripts (splice variants), 75 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ly6g6e-201 ENSMUST00000013910.4 1142 134aa ENSMUSP00000013910.4 Protein coding CCDS28679 Q8K1T6 TSL:1 GENCODE basic APPRIS P2 Ly6g6e-203 ENSMUST00000172678.7 750 134aa ENSMUSP00000134073.1 Protein coding CCDS28679 Q8K1T6 TSL:3 GENCODE basic APPRIS P2 Ly6g6e-204 ENSMUST00000172959.1 501 166aa ENSMUSP00000133753.1 Protein coding - Q8K1T6 TSL:1 GENCODE basic APPRIS ALT2 Ly6g6e-202 ENSMUST00000172494.7 464 107aa ENSMUSP00000133645.1 Protein coding - G3UXD4 TSL:3 GENCODE basic Page 6 of 8 https://www.alphaknockout.com 21.90 kb Forward strand 35.070Mb 35.075Mb 35.080Mb 35.085Mb Genes (Comprehensive set... Ly6g6c-201 >protein coding Ly6g6e-202 >protein coding Ly6g6c-202 >protein coding Ly6g6e-203 >protein coding Ly6g6c-203 >protein coding Ly6g6e-201 >protein coding Ly6g6e-204 >protein coding Contigs AC087117.9 > Genes < Ly6g6d-201protein coding < Ly6g6f-201protein coding (Comprehensive set... < Ly6g6d-202lncRNA < Ly6g6d-204retained intron < Ly6g6d-205retained intron < Ly6g6d-203lncRNA Regulatory Build 35.070Mb 35.075Mb 35.080Mb 35.085Mb Reverse strand 21.90 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000013910 1.87 kb Forward strand Ly6g6e-201 >protein coding ENSMUSP00000013... Low complexity (Seg) Cleavage site (Sign... Superfamily SSF57302 Pfam Activin types I and II receptor domain PANTHER Lymphocyte antigen 6G6e Gene3D 2.10.60.10 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) M Y M Y Variant Legend frameshift variant missense variant synonymous variant Scale bar 0 20 40 60 80 100 134 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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