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Mouse Rtl6 Knockout Project (CRISPR/Cas9)

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Mouse Rtl6 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rtl6 Knockout Project (CRISPR/Cas9) Objective: To create a Rtl6 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rtl6 gene (NCBI Reference Sequence: NM_177630.3 ; Ensembl: ENSMUSG00000055745 ) is located on Mouse chromosome 15. 1 exon is identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 1 (Transcript: ENSMUST00000069476). Exon 1 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 1 starts from about 0.14% of the coding region. Exon 1 covers 100.0% of the coding region. The size of effective KO region: ~727 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 Legends Exon of mouse Rtl6 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 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.0% 480) | C(28.1% 562) | T(22.0% 440) | G(25.9% 518) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(20.35% 407) | C(27.7% 554) | T(24.6% 492) | G(27.35% 547) 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 8 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% chr15 - 84557194 84559193 2000 browser details YourSeq 75 659 936 2000 96.3% chr18 + 32560466 32560760 295 browser details YourSeq 57 629 707 2000 98.4% chr7 - 109643550 109643645 96 browser details YourSeq 55 659 807 2000 96.7% chr1 - 89476539 89476726 188 browser details YourSeq 51 63 136 2000 88.5% chr15 - 86998536 86998610 75 browser details YourSeq 51 654 708 2000 98.2% chr17 + 75465673 75465734 62 browser details YourSeq 50 659 708 2000 100.0% chr9 - 47825024 47825073 50 browser details YourSeq 50 659 708 2000 100.0% chr4 - 130953971 130954020 50 browser details YourSeq 50 659 708 2000 100.0% chr3 + 41137228 41137277 50 browser details YourSeq 50 659 708 2000 100.0% chr17 + 64908013 64908062 50 browser details YourSeq 50 659 710 2000 98.1% chr1 + 122666095 122666146 52 browser details YourSeq 49 659 707 2000 100.0% chr8 - 118181581 118181629 49 browser details YourSeq 49 659 707 2000 100.0% chr14 + 44213703 44213751 49 browser details YourSeq 49 659 707 2000 100.0% chr1 + 187919974 187920022 49 browser details YourSeq 48 659 708 2000 98.0% chr4 - 98356066 98356115 50 browser details YourSeq 48 657 708 2000 98.0% chr4 + 10586712 10586763 52 browser details YourSeq 48 659 708 2000 98.0% chr16 + 91306779 91306828 50 browser details YourSeq 48 659 708 2000 98.0% chr1 + 88400424 88400473 50 browser details YourSeq 47 659 708 2000 91.7% chr5 - 96500171 96500218 48 browser details YourSeq 47 659 705 2000 100.0% chr3 - 117957161 117957207 47 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% chr15 - 84554465 84556464 2000 browser details YourSeq 34 624 660 2000 97.3% chr1 - 88253723 88254169 447 browser details YourSeq 32 621 701 2000 94.5% chr2 + 83755615 83755696 82 browser details YourSeq 29 1619 1651 2000 83.9% chr9 - 69799611 69799641 31 browser details YourSeq 27 1243 1282 2000 86.7% chr12 - 110265035 110265072 38 browser details YourSeq 26 619 645 2000 100.0% chr1 + 58213723 58213752 30 browser details YourSeq 22 619 641 2000 100.0% chrX - 139377328 139377351 24 browser details YourSeq 21 621 643 2000 95.7% chr1 - 24503131 24503153 23 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Rtl6 retrotransposon Gag like 6 [ Mus musculus (house mouse) ] Gene ID: 223732, updated on 26-Jun-2020 Gene summary Official Symbol Rtl6 provided by MGI Official Full Name retrotransposon Gag like 6 provided by MGI Primary source MGI:MGI:2675858 See related Ensembl:ENSMUSG00000055745 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 Mar6; Mart6; Ldoc1l; BC058638 Orthologs human all Genomic context Location: 15; 15 E2 See Rtl6 in Genome Data Viewer Exon count: 1 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (84553398..84557823, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (84383828..84388253, complement) Chromosome 15 - NC_000081.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Rtl6 ENSMUSG00000055745 Description retrotransposon Gag like 6 [Source:MGI Symbol;Acc:MGI:2675858] Gene Synonyms Ldoc1l, Mar6, Mart6 Location Chromosome 15: 84,553,398-84,557,823 reverse strand. GRCm38:CM001008.2 About this gene This gene has 1 transcript (splice variant), 84 orthologues, 8 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rtl6-201 ENSMUST00000069476.4 4426 243aa ENSMUSP00000069947.4 Protein coding CCDS37168 Q505G4 TSL:NA GENCODE basic APPRIS P1 24.43 kb Forward strand 84.545Mb 84.550Mb 84.555Mb 84.560Mb 84.565Mb Contigs AL611986.15 > Genes (Comprehensive set... < Rtl6-201protein coding Regulatory Build 84.545Mb 84.550Mb 84.555Mb 84.560Mb 84.565Mb Reverse strand 24.43 kb Regulation Legend Enhancer Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000069476 < Rtl6-201protein coding Reverse strand 4.43 kb ENSMUSP00000069... MobiDB lite Coiled-coils (Ncoils) Pfam Domain of unknown function DUF4939 PANTHER PTHR15503:SF5 LDOC1-related All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 243 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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