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Mouse Letmd1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Letmd1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Letmd1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Letmd1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Letmd1 gene (NCBI Reference Sequence: NM_134093 ; Ensembl: ENSMUSG00000037353 ) is located on Mouse chromosome 15. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000037001). Exon 5~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Letmd1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-81M8 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 5 starts from about 43.89% of the coding region. The knockout of Exon 5~7 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2391 bp, and the size of intron 7 for 3'-loxP site insertion: 1135 bp. The size of effective cKO region: ~1132 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 5 6 7 8 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Letmd1 Homology arm cKO region 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(7632bp) | A(24.03% 1834) | C(24.33% 1857) | T(26.85% 2049) | G(24.79% 1892) 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% chr15 + 100471774 100474773 3000 browser details YourSeq 171 540 1105 3000 85.6% chr13 - 38597991 38598435 445 browser details YourSeq 169 534 1104 3000 83.3% chr4 + 134033511 134033836 326 browser details YourSeq 156 541 1102 3000 83.9% chr7 + 139954544 139954829 286 browser details YourSeq 149 492 655 3000 93.9% chr10 + 20313370 20313531 162 browser details YourSeq 146 494 653 3000 96.3% chr10 - 59997296 59997495 200 browser details YourSeq 143 540 1491 3000 78.6% chrX - 8003186 8003498 313 browser details YourSeq 127 544 1083 3000 79.4% chr5 + 13955097 13955390 294 browser details YourSeq 125 534 1104 3000 93.8% chr7 - 28354944 28355693 750 browser details YourSeq 123 573 1601 3000 75.8% chr4 + 109937771 109938117 347 browser details YourSeq 109 1013 1490 3000 88.8% chr10 - 91166222 91331032 164811 browser details YourSeq 104 541 652 3000 96.5% chr15 + 101281441 101281552 112 browser details YourSeq 95 516 620 3000 95.3% chr9 - 55243764 55243868 105 browser details YourSeq 94 545 640 3000 99.0% chr3 - 9175224 9175319 96 browser details YourSeq 94 1062 1490 3000 79.2% chr12 - 87404190 87404591 402 browser details YourSeq 92 548 648 3000 96.1% chr19 - 3677929 3678043 115 browser details YourSeq 92 534 635 3000 95.1% chr1 - 60134685 60134786 102 browser details YourSeq 86 998 1101 3000 92.3% chr18 + 32935540 32935660 121 browser details YourSeq 85 1372 1491 3000 85.9% chr16 + 36809368 36809489 122 browser details YourSeq 83 619 1105 3000 88.8% chr19 - 37542556 37543096 541 Note: The 3000 bp section upstream of Exon 5 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% chr15 + 100475906 100478905 3000 browser details YourSeq 60 1254 1340 3000 92.9% chr10 + 64723803 64723908 106 browser details YourSeq 56 1250 1344 3000 87.5% chr12 + 7309435 7309526 92 browser details YourSeq 49 1250 1321 3000 93.0% chr10 - 129121212 129121288 77 browser details YourSeq 45 1084 1168 3000 94.2% chr9 + 54922423 54922510 88 browser details YourSeq 44 1116 1347 3000 65.6% chr12 + 87188361 87188518 158 browser details YourSeq 44 1109 1194 3000 87.1% chr11 + 67474157 67474241 85 browser details YourSeq 43 1281 1340 3000 94.0% chr10 + 125119503 125119650 148 browser details YourSeq 42 1121 1182 3000 83.4% chr5 - 136583259 136583316 58 browser details YourSeq 40 1094 1166 3000 87.5% chr10 - 78168405 78168476 72 browser details YourSeq 40 314 553 3000 67.5% chr2 + 83622038 83622188 151 browser details YourSeq 39 1074 1186 3000 91.5% chr10 - 41492515 41492627 113 browser details YourSeq 39 1135 1185 3000 93.2% chr8 + 37681846 37681896 51 browser details YourSeq 37 1112 1189 3000 85.4% chr1 - 171962262 171962336 75 browser details YourSeq 37 1080 1186 3000 87.5% chr12 + 39408766 39408872 107 browser details YourSeq 36 1080 1168 3000 87.5% chr11 - 85246797 85246886 90 browser details YourSeq 35 1080 1185 3000 87.3% chr10 - 125119801 125119906 106 browser details YourSeq 34 1135 1186 3000 94.8% chrX + 166561455 166561506 52 browser details YourSeq 33 17 59 3000 88.4% chr10 - 22518166 22518208 43 browser details YourSeq 33 1135 1185 3000 92.4% chr11 + 47000066 47000116 51 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Letmd1 LETM1 domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 68614, updated on 24-Oct-2019 Gene summary Official Symbol Letmd1 provided by MGI Official Full Name LETM1 domain containing 1 provided by MGI Primary source MGI:MGI:1915864 See related Ensembl:ENSMUSG00000037353 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 Mccr; HCCR1; HCCR-2; MCC-32; AI593524; BB130465; BB235638; 1110019O13Rik Expression Ubiquitous expression in adrenal adult (RPKM 33.3), mammary gland adult (RPKM 24.6) and 28 other tissues See more Orthologs human all Genomic context Location: 15; 15 F1 See Letmd1 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (100462421..100479252) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (100299465..100309683) Chromosome 15 - NC_000081.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Letmd1 ENSMUSG00000037353 Description LETM1 domain containing 1 [Source:MGI Symbol;Acc:MGI:1915864] Gene Synonyms 1110019O13Rik, HCCR-2, HCCR1 Location Chromosome 15: 100,469,023-100,479,164 forward strand. GRCm38:CM001008.2 About this gene This gene has 10 transcripts (splice variants), 197 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Letmd1- ENSMUST00000037001.9 2502 360aa ENSMUSP00000037546.8 Protein coding CCDS27838 Q924L1 TSL:1 201 GENCODE basic APPRIS P2 Letmd1- ENSMUST00000230294.1 2266 203aa ENSMUSP00000155807.1 Protein coding - Q924L1 GENCODE 207 basic Letmd1- ENSMUST00000229648.1 1222 271aa ENSMUSP00000155084.1 Protein coding - Q924L1 GENCODE 206 basic APPRIS ALT2 Letmd1- ENSMUST00000229012.1 2389 123aa ENSMUSP00000155243.1 Nonsense mediated - A0A2R8VK53 - 202 decay Letmd1- ENSMUST00000229596.1 2917 No - Retained intron - - - 205 protein Letmd1- ENSMUST00000231001.1 2153 No - Retained intron - - - 210 protein Letmd1- ENSMUST00000230579.1 747 No - Retained intron - - - 209 protein Letmd1- ENSMUST00000229457.1 630 No - Retained intron - - - 204 protein Letmd1- ENSMUST00000230339.1 630 No - Retained intron - - - 208 protein Letmd1- ENSMUST00000229372.1 497 No - Retained intron - - - 203 protein Page 6 of 8 https://www.alphaknockout.com 30.14 kb Forward strand 100.46Mb 100.47Mb 100.48Mb Genes (Comprehensive set... Letmd1-201 >protein coding Letmd1-207 >protein coding Letmd1-208 >retained intron Letmd1-204 >retained intron Letmd1-206 >protein coding Letmd1-202 >nonsense mediated decay Letmd1-205 >retained intron Letmd1-210 >retained intron Letmd1-209 >retained intron Letmd1-203 >retained intron Contigs < AC139571.2 Genes < 5330439K02Rik-201lncRNA < Csrnp2-201protein coding (Comprehensive set... Regulatory Build 100.46Mb 100.47Mb 100.48Mb Reverse strand 30.14 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000037001 10.14 kb Forward strand Letmd1-201 >protein coding ENSMUSP00000037... Transmembrane heli... Low complexity (Seg) Pfam LETM1-like PROSITE profiles Letm1 ribosome-binding domain PANTHER PTHR14009:SF13 PTHR14009 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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