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Mouse Elovl5 Knockout Project (CRISPR/Cas9)

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Mouse Elovl5 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Elovl5 Knockout Project (CRISPR/Cas9) Objective: To create a Elovl5 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Elovl5 gene (NCBI Reference Sequence: NM_134255 ; Ensembl: ENSMUSG00000032349 ) is located on Mouse chromosome 9. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000034904). Exon 3 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 homozygous for a gene trapped allele have defects in fatty acid synthesis in the liver that result in hepatic steatosis. Also, majority of female mice have defects in female fertility. Exon 3 starts from about 6.58% of the coding region. Exon 3 covers 20.96% of the coding region. The size of effective KO region: ~188 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 8 Legends Exon of mouse Elovl5 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 Exon 3 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 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. 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(26.8% 536) | C(21.1% 422) | T(27.45% 549) | G(24.65% 493) Note: The 2000 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(2000bp) | A(23.35% 467) | C(20.45% 409) | T(31.65% 633) | G(24.55% 491) Note: The 2000 bp section downstream 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. 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% chr9 + 77958867 77960866 2000 browser details YourSeq 148 1292 1512 2000 85.8% chr12 - 11180548 11180773 226 browser details YourSeq 140 1298 1511 2000 84.1% chr11 + 19672464 19672661 198 browser details YourSeq 136 1162 1512 2000 79.9% chr8 - 70324582 70324769 188 browser details YourSeq 135 1297 1504 2000 84.8% chr13 - 47988568 47988771 204 browser details YourSeq 128 1298 1504 2000 83.2% chr1 - 53348828 53349016 189 browser details YourSeq 123 1289 1514 2000 84.3% chr2 - 129654523 129654738 216 browser details YourSeq 121 1341 1516 2000 90.1% chr1 - 93126536 93126713 178 browser details YourSeq 115 1351 1509 2000 86.3% chr10 - 112106521 112106678 158 browser details YourSeq 114 1298 1514 2000 86.4% chr8 - 121495211 121495424 214 browser details YourSeq 114 1346 1508 2000 87.0% chr7 - 77533942 77534106 165 browser details YourSeq 112 1297 1513 2000 88.8% chr7 - 46522445 46522663 219 browser details YourSeq 112 1331 1517 2000 87.5% chr1 - 170500191 170500578 388 browser details YourSeq 111 1293 1484 2000 92.4% chr10 + 4020760 4021138 379 browser details YourSeq 110 1300 1461 2000 88.4% chr13 - 107167620 107167785 166 browser details YourSeq 110 1301 1512 2000 80.5% chr1 - 63503118 63503297 180 browser details YourSeq 110 1347 1504 2000 87.1% chr9 + 55046414 55046574 161 browser details YourSeq 109 1297 1508 2000 93.0% chr8 - 25046595 25046807 213 browser details YourSeq 109 1299 1484 2000 87.8% chr1 + 134263094 134263278 185 browser details YourSeq 108 1157 1469 2000 79.3% chr8 - 126902239 126902428 190 Note: The 2000 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 2000 1 2000 2000 100.0% chr9 + 77961055 77963054 2000 browser details YourSeq 46 1162 1251 2000 92.6% chr1 + 56722212 56722626 415 browser details YourSeq 42 1158 1207 2000 95.7% chr2 - 106418540 106418591 52 browser details YourSeq 37 1171 1212 2000 97.6% chr1 - 15235503 15235547 45 browser details YourSeq 35 1161 1208 2000 90.7% chr1 - 96843520 96843577 58 browser details YourSeq 31 1164 1201 2000 94.5% chr1 - 55899120 55899157 38 browser details YourSeq 31 1169 1201 2000 97.0% chr4 + 99623464 99623496 33 browser details YourSeq 25 653 680 2000 84.7% chr3 - 90165551 90165576 26 browser details YourSeq 25 1178 1203 2000 100.0% chr1 - 63075835 63075863 29 browser details YourSeq 23 1169 1192 2000 100.0% chr1 - 123023917 123023941 25 browser details YourSeq 23 1467 1489 2000 100.0% chr6 + 60565534 60565556 23 browser details YourSeq 22 1366 1387 2000 100.0% chr11 - 85909241 85909262 22 browser details YourSeq 22 1297 1319 2000 100.0% chr1 - 13450814 13450838 25 browser details YourSeq 22 1297 1319 2000 100.0% chr1 + 89224032 89224056 25 browser details YourSeq 21 1276 1296 2000 100.0% chr11 + 12190291 12190311 21 browser details YourSeq 20 1476 1495 2000 100.0% chr1 - 96353522 96353541 20 browser details YourSeq 20 1228 1247 2000 100.0% chr15 + 88774775 88774794 20 browser details YourSeq 20 1177 1196 2000 100.0% chr1 + 82719502 82719521 20 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Elovl5 ELOVL family member 5, elongation of long chain fatty acids (yeast) [ Mus musculus (house mouse) ] Gene ID: 68801, updated on 12-Aug-2019 Gene summary Official Symbol Elovl5 provided by MGI Official Full Name ELOVL family member 5, elongation of long chain fatty acids (yeast) provided by MGI Primary source MGI:MGI:1916051 See related Ensembl:ENSMUSG00000032349 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 HELO1; AI747313; AU043003; 1110059L23Rik Expression Broad expression in liver adult (RPKM 103.9), adrenal adult (RPKM 79.4) and 25 other tissues See more Orthologs human all Genomic context Location: 9; 9 E1 See Elovl5 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (77917365..77984519) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (77765172..77832326) Chromosome 9 - NC_000075.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Elovl5 ENSMUSG00000032349 Description ELOVL family member 5, elongation of long chain fatty acids (yeast) [Source:MGI Symbol;Acc:MGI:1916051] Gene Synonyms 1110059L23Rik Location Chromosome 9: 77,917,364-77,984,519 forward strand. GRCm38:CM001002.2 About this gene This gene has 4 transcripts (splice variants), 203 orthologues, 6 paralogues, 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 Elovl5-201 ENSMUST00000034904.13 2782 299aa ENSMUSP00000034904.7 Protein coding CCDS23355 Q8BHI7 TSL:1 GENCODE basic APPRIS P1 Elovl5-202 ENSMUST00000133757.7 638 180aa ENSMUSP00000123121.1 Protein coding - D3Z136 CDS 3' incomplete TSL:3 Elovl5-203 ENSMUST00000134072.1 346 71aa ENSMUSP00000120171.1 Protein coding - D3Z0Z6 CDS 3' incomplete TSL:3 Elovl5-204 ENSMUST00000148398.1 685 No protein - Retained intron - - TSL:1 87.16 kb Forward strand Genes (Comprehensive set... Elovl5-201 >protein coding Elovl5-204 >retained intron Gm34829-202 >lncRNA Elovl5-202 >protein coding Gm34829-201 >lncRNA Elovl5-203 >protein coding Contigs < AC159886.2 AC160334.2 > Regulatory Build Reverse strand 87.16 kb Regulation Legend Enhancer 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: ENSMUST00000034904 67.16 kb Forward strand Elovl5-201 >protein coding ENSMUSP00000034... Transmembrane heli... Low complexity (Seg) Pfam ELO family PANTHER Elongation of very long chain fatty acids protein 5 ELO family HAMAP Elongation of very long chain fatty acids protein 5 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 299 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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