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Mouse Erlin2 Knockout Project (CRISPR/Cas9)

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Mouse Erlin2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Erlin2 Knockout Project (CRISPR/Cas9) Objective: To create a Erlin2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Erlin2 gene (NCBI Reference Sequence: NM_153592 ; Ensembl: ENSMUSG00000031483 ) is located on Mouse chromosome 8. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000033873). Exon 2~10 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 2 starts from the coding region. Exon 2~10 covers 72.45% of the coding region. The size of effective KO region: ~8419 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 9 10 12 Legends Exon of mouse Erlin2 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1150 bp section upstream of Exon 2 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 10 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 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(1150bp) | A(22.09% 254) | C(26.26% 302) | T(24.43% 281) | G(27.22% 313) Note: The 1150 bp section upstream of Exon 2 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(27.2% 544) | C(23.65% 473) | T(23.9% 478) | G(25.25% 505) Note: The 2000 bp section downstream of Exon 10 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 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 1150 1 1150 1150 100.0% chr8 + 27023923 27025072 1150 browser details YourSeq 38 625 667 1150 95.4% chr1 - 32359005 32359048 44 browser details YourSeq 30 625 664 1150 90.7% chr5 + 107493676 107493714 39 browser details YourSeq 30 625 660 1150 91.7% chr10 + 39995956 39995991 36 browser details YourSeq 25 405 429 1150 100.0% chr12 + 79820742 79820766 25 browser details YourSeq 25 403 427 1150 100.0% chr11 + 120482355 120482379 25 browser details YourSeq 23 550 575 1150 96.0% chr1 - 78731601 78731637 37 browser details YourSeq 22 403 424 1150 100.0% chr16 - 93291362 93291383 22 browser details YourSeq 22 629 650 1150 100.0% chr1 - 65074327 65074348 22 browser details YourSeq 21 409 429 1150 100.0% chr12 - 72657961 72657981 21 browser details YourSeq 21 403 423 1150 100.0% chr1 + 171174650 171174670 21 browser details YourSeq 20 797 816 1150 100.0% chr1 + 122607762 122607781 20 Note: The 1150 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 2000 1 2000 2000 100.0% chr8 + 27033477 27035476 2000 browser details YourSeq 141 176 324 2000 97.4% chr8 + 27034850 27034998 149 browser details YourSeq 141 1374 1522 2000 97.4% chr8 + 27033652 27033800 149 browser details YourSeq 85 1581 1766 2000 79.4% chr5 - 136172127 136172298 172 browser details YourSeq 56 1586 1700 2000 93.8% chr15 - 31245553 31245678 126 browser details YourSeq 56 1558 1699 2000 81.6% chr2 + 121477093 121477223 131 browser details YourSeq 47 1587 1686 2000 87.8% chr1 - 52241120 52241218 99 browser details YourSeq 44 1655 1702 2000 98.0% chr8 + 3687300 3687388 89 browser details YourSeq 42 1580 1702 2000 90.2% chr11 + 92156160 92156283 124 browser details YourSeq 40 1228 1347 2000 95.5% chr11 - 112890822 112891085 264 browser details YourSeq 39 1223 1339 2000 86.8% chr3 - 64976679 64976796 118 browser details YourSeq 39 298 351 2000 86.8% chr11 + 115636517 115636589 73 browser details YourSeq 38 1574 1631 2000 93.1% chr6 - 87133709 87133766 58 browser details YourSeq 38 1559 1621 2000 73.9% chr15 - 9349379 9349429 51 browser details YourSeq 37 1613 1701 2000 90.3% chr2 - 121518825 121518912 88 browser details YourSeq 36 1650 1691 2000 92.9% chr9 + 40830365 40830406 42 browser details YourSeq 35 1650 1702 2000 83.1% chr11 - 100162690 100162742 53 browser details YourSeq 31 1670 1702 2000 97.0% chr6 - 71003599 71003631 33 browser details YourSeq 31 1670 1702 2000 97.0% chr14 + 47436321 47436353 33 browser details YourSeq 30 379 410 2000 100.0% chr2 - 49390346 49390386 41 Note: The 2000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Erlin2 ER lipid raft associated 2 [ Mus musculus (house mouse) ] Gene ID: 244373, updated on 24-Oct-2019 Gene summary Official Symbol Erlin2 provided by MGI Official Full Name ER lipid raft associated 2 provided by MGI Primary source MGI:MGI:2387215 See related Ensembl:ENSMUSG00000031483 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 Spfh2; C87251; BC036333 Expression Ubiquitous expression in kidney adult (RPKM 24.1), genital fat pad adult (RPKM 18.2) and 28 other tissues See more Orthologs human all Genomic context Location: 8; 8 A2 See Erlin2 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (27022408..27039437) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (28134331..28149896) Chromosome 8 - NC_000074.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Erlin2 ENSMUSG00000031483 Description ER lipid raft associated 2 [Source:MGI Symbol;Acc:MGI:2387215] Gene Synonyms Spfh2 Location Chromosome 8: 27,023,261-27,040,328 forward strand. GRCm38:CM001001.2 About this gene This gene has 9 transcripts (splice variants), 195 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Erlin2- ENSMUST00000033873.8 4838 340aa ENSMUSP00000033873.7 Protein coding CCDS22209 Q8BFZ9 TSL:1 201 GENCODE basic APPRIS P1 Erlin2- ENSMUST00000209563.1 965 185aa ENSMUSP00000147310.1 Protein coding - A0A1B0GQZ0 CDS 3' 204 incomplete TSL:3 Erlin2- ENSMUST00000209520.1 940 220aa ENSMUSP00000147897.1 Protein coding - A0A1B0GSD8 CDS 3' 203 incomplete TSL:5 Erlin2- ENSMUST00000209795.1 833 150aa ENSMUSP00000148192.1 Protein coding - A0A1B0GT43 CDS 3' 205 incomplete TSL:5 Erlin2- ENSMUST00000209504.1 752 251aa ENSMUSP00000148228.1 Protein coding - A0A1B0GT70 CDS 5' and 3' 202 incomplete TSL:3 Erlin2- ENSMUST00000211043.1 686 159aa ENSMUSP00000147616.1 Protein coding - A0A1B0GRQ1 CDS 3' 208 incomplete TSL:3 Erlin2- ENSMUST00000209976.1 554 85aa ENSMUSP00000147516.1 Protein coding - A0A1B0GRG7 CDS 3' 206 incomplete TSL:3 Erlin2- ENSMUST00000211233.1 326 36aa ENSMUSP00000147642.1 Nonsense mediated - A0A1B0GRS4 CDS 5' 209 decay incomplete TSL:3 Erlin2- ENSMUST00000210445.1 345 No - Retained intron - - TSL:2 207 protein Page 7 of 9 https://www.alphaknockout.com 37.07 kb Forward strand 27.02Mb 27.03Mb 27.04Mb 27.05Mb Genes (Comprehensive set... Erlin2-205 >protein coding Plpbp-206 >nonsense mediated decay Erlin2-206 >protein coding Erlin2-202 >protein coding Plpbp-201 >protein coding Erlin2-201 >protein coding Plpbp-203 >protein coding Erlin2-208 >protein coding Plpbp-205 >protein coding Erlin2-204 >protein coding Plpbp-202 >protein coding Erlin2-203 >protein coding Plpbp-211 >protein coding Erlin2-207 >retained intron Plpbp-213 >protein coding Erlin2-209 >nonsense mediated decay Plpbp-212 >lncRNA Plpbp-204 >lncRNA Plpbp-210 >lncRNA Plpbp-208 >protein coding Plpbp-207 >retained intron Plpbp-209 >retained intron Contigs < AC115820.11 Genes < Gm19164-201processed pseudogene < Proscos-202lncRNA (Comprehensive set... < Proscos-201lncRNA Regulatory Build 27.02Mb 27.03Mb 27.04Mb 27.05Mb Reverse strand 37.07 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000033873 16.53 kb Forward strand Erlin2-201 >protein coding ENSMUSP00000033... Low complexity (Seg) Cleavage site (Sign... SMART Band 7 domain Pfam Band 7 domain PANTHER Erlin1/2 PTHR15351:SF4 CDD Erlin1/2 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 340 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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