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Mouse Cyb5r1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Cyb5r1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cyb5r1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Cyb5r1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cyb5r1 gene (NCBI Reference Sequence: NM_028057 ; Ensembl: ENSMUSG00000026456 ) is located on Mouse chromosome 1. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000027726). Exon 6~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cyb5r1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-260A4 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 6 starts from about 52.02% of the coding region. The knockout of Exon 6~7 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 700 bp, and the size of intron 7 for 3'-loxP site insertion: 806 bp. The size of effective cKO region: ~1783 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 4 5 6 7 8 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Cyb5r1 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(8283bp) | A(24.68% 2044) | C(24.31% 2014) | T(27.2% 2253) | G(23.81% 1972) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chr1 + 134405155 134408154 3000 browser details YourSeq 439 1 500 3000 99.2% chr4 + 137386553 137387176 624 browser details YourSeq 432 1 439 3000 99.6% chr16 + 81752638 81753076 439 browser details YourSeq 429 1 437 3000 98.7% chr6 - 134009208 134009643 436 browser details YourSeq 429 2 449 3000 98.7% chr10 - 19379856 19380311 456 browser details YourSeq 429 1 438 3000 99.6% chr1 - 171857158 171857596 439 browser details YourSeq 428 1 437 3000 99.6% chr10 - 24325946 24326382 437 browser details YourSeq 428 1 437 3000 99.4% chr14 + 87391687 87392123 437 browser details YourSeq 427 1 438 3000 99.4% chr6 + 127520533 127520970 438 browser details YourSeq 426 1 437 3000 99.1% chr11 - 15167920 15168356 437 browser details YourSeq 426 1 437 3000 99.4% chr6 + 115829355 115829791 437 browser details YourSeq 426 1 437 3000 99.4% chr1 + 7481661 7482097 437 browser details YourSeq 425 2 438 3000 99.1% chrX - 16339324 16339761 438 browser details YourSeq 425 5 438 3000 99.4% chr6 - 134166978 134167411 434 browser details YourSeq 425 1 438 3000 99.1% chr6 - 124797029 124797466 438 browser details YourSeq 425 1 437 3000 98.9% chr5 - 89203777 89204219 443 browser details YourSeq 425 1 438 3000 99.1% chrY + 40111379 40111816 438 browser details YourSeq 425 1 438 3000 99.1% chr1 + 111696290 111696727 438 browser details YourSeq 424 1 437 3000 98.9% chr6 - 108082376 108082812 437 browser details YourSeq 424 1 438 3000 98.0% chr3 - 53113418 53113854 437 Note: The 3000 bp section upstream of Exon 6 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% chr1 + 134409938 134412937 3000 browser details YourSeq 225 2505 2979 3000 91.5% chr7 - 116073492 116271460 197969 browser details YourSeq 191 2489 2978 3000 88.7% chr11 + 62469947 62470566 620 browser details YourSeq 184 2489 2982 3000 83.7% chrX + 73735497 73735806 310 browser details YourSeq 154 2576 2974 3000 92.9% chr7 + 3172873 3173485 613 browser details YourSeq 149 45 275 3000 87.5% chr13 + 38041741 38042408 668 browser details YourSeq 149 2597 2982 3000 83.3% chr1 + 179565111 179565412 302 browser details YourSeq 147 2633 2978 3000 81.9% chrX - 151404323 151404529 207 browser details YourSeq 147 2576 2979 3000 82.9% chr4 - 126188686 126188885 200 browser details YourSeq 143 2597 2984 3000 91.0% chr2 - 174595271 174792182 196912 browser details YourSeq 142 2483 2963 3000 87.0% chr12 - 59057375 59057998 624 browser details YourSeq 142 2818 2992 3000 89.9% chr6 + 46077114 46077285 172 browser details YourSeq 141 2580 2983 3000 81.3% chr17 + 46749090 46749287 198 browser details YourSeq 138 2813 2997 3000 90.2% chr15 + 100344819 100345004 186 browser details YourSeq 138 2820 2983 3000 90.6% chr14 + 85826119 85826277 159 browser details YourSeq 137 2634 2979 3000 81.3% chr3 - 68876172 68876339 168 browser details YourSeq 137 2817 2974 3000 93.7% chr2 + 17834727 17834914 188 browser details YourSeq 137 2819 2978 3000 91.7% chr10 + 77627999 77628156 158 browser details YourSeq 136 2818 2983 3000 91.6% chr11 - 12764938 12765107 170 browser details YourSeq 136 2818 2983 3000 91.6% chr2 + 104105200 104105368 169 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: Cyb5r1 cytochrome b5 reductase 1 [ Mus musculus (house mouse) ] Gene ID: 72017, updated on 12-Aug-2019 Gene summary Official Symbol Cyb5r1 provided by MGI Official Full Name cytochrome b5 reductase 1 provided by MGI Primary source MGI:MGI:1919267 See related Ensembl:ENSMUSG00000026456 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 B5R.1; C80155; Nqo3a2; 1500005G05Rik Expression Ubiquitous expression in adrenal adult (RPKM 21.2), ovary adult (RPKM 17.0) and 28 other tissues See more Orthologs human all Genomic context Location: 1; 1 E4 See Cyb5r1 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (134405781..134411738) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (136302567..136308315) Chromosome 1 - NC_000067.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Cyb5r1 ENSMUSG00000026456 Description cytochrome b5 reductase 1 [Source:MGI Symbol;Acc:MGI:1919267] Gene Synonyms 1500005G05Rik, B5R.1, Nqo3a2 Location Chromosome 1: 134,405,559-134,411,740 forward strand. GRCm38:CM000994.2 About this gene This gene has 10 transcripts (splice variants), 109 orthologues, 5 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cyb5r1- ENSMUST00000027726.13 1837 305aa ENSMUSP00000027726.7 Protein coding CCDS15308 Q9DB73 TSL:1 201 GENCODE basic APPRIS P1 Cyb5r1- ENSMUST00000173908.7 563 176aa ENSMUSP00000134488.1 Protein coding - G3UZG6 CDS 3' 208 incomplete TSL:5 Cyb5r1- ENSMUST00000154237.7 2420 228aa ENSMUSP00000133385.1 Nonsense mediated - Q9DB73 TSL:1 205 decay Cyb5r1- ENSMUST00000172795.1 720 80aa ENSMUSP00000133842.1 Nonsense mediated - G3UXV8 CDS 5' 207 decay incomplete TSL:5 Cyb5r1- ENSMUST00000127412.8 530 100aa ENSMUSP00000134354.1 Nonsense mediated - G3UZ57 CDS 5' 202 decay incomplete TSL:3 Cyb5r1- ENSMUST00000133686.7 2341 No - Retained intron - - TSL:1 204 protein Cyb5r1- ENSMUST00000172496.7 957 No - Retained intron - - TSL:2 206 protein Cyb5r1- ENSMUST00000186489.6 875 No - lncRNA - - TSL:1 210 protein Cyb5r1- ENSMUST00000130836.7 434 No - lncRNA - - TSL:5 203 protein Cyb5r1- ENSMUST00000174315.7 338 No - lncRNA - - TSL:3 209 protein Page 6 of 8 https://www.alphaknockout.com 26.18 kb Forward strand 134.40Mb 134.41Mb 134.42Mb Genes (Comprehensive set... Cyb5r1-210 >lncRNA Adipor1-201 >protein coding Cyb5r1-201 >protein coding Adipor1-202 >protein coding Cyb5r1-205 >nonsense mediated decay Adipor1-204 >lncRNA Cyb5r1-209 >lncRNA Adipor1-203 >retained intron Cyb5r1-203 >lncRNA Cyb5r1-208 >protein coding Cyb5r1-206 >retained intron Cyb5r1-204 >retained intron Cyb5r1-207 >nonsense mediated decay Cyb5r1-202 >nonsense mediated decay Contigs AC131592.10 > Regulatory Build 134.40Mb 134.41Mb 134.42Mb Reverse strand 26.18 kb Regulation Legend CTCF Promoter Promoter Flank 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: ENSMUST00000027726 5.96 kb Forward strand Cyb5r1-201 >protein coding ENSMUSP00000027... Transmembrane heli... Low complexity (Seg) Superfamily Ferredoxin-NADP
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