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Mouse Srxn1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Srxn1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Srxn1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Srxn1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Srxn1 gene (NCBI Reference Sequence: NM_029688 ; Ensembl: ENSMUSG00000032802 ) is located on Mouse chromosome 2. 2 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000041500). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Srxn1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-214I20 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: Mice homozygous for a knock-out allele exhibit increased sensitivity to LPS-induced shock. Exon 1 covers 56.77% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of intron 1 for 3'-loxP site insertion: 3041 bp. The size of effective cKO region: ~524 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele A gRNA region T 5' G 3' 1 2 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Srxn1 cKO region loxP site Page 2 of 7 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(6764bp) | A(23.23% 1571) | C(26.02% 1760) | T(25.46% 1722) | G(25.3% 1711) 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 7 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% chr2 + 152102685 152105684 3000 browser details YourSeq 379 176 1772 3000 85.8% chr9 + 71745084 71823233 78150 browser details YourSeq 336 1359 1985 3000 88.3% chr6 + 37778589 37779322 734 browser details YourSeq 326 1374 2034 3000 83.5% chr14 - 54105524 54106041 518 browser details YourSeq 314 1374 1942 3000 85.7% chr4 - 143331416 143332207 792 browser details YourSeq 309 1359 2034 3000 88.9% chr10 - 53681943 53719288 37346 browser details YourSeq 289 1495 1983 3000 87.0% chrX - 105045391 105046104 714 browser details YourSeq 286 1380 1948 3000 86.3% chr17 - 7859336 7860135 800 browser details YourSeq 286 1335 2033 3000 82.1% chr8 + 45415823 45416347 525 browser details YourSeq 283 1374 2034 3000 81.8% chr13 + 49404001 49404535 535 browser details YourSeq 281 1312 1756 3000 85.0% chr8 - 95049691 95050299 609 browser details YourSeq 281 1358 2034 3000 81.3% chr13 - 64564857 64565388 532 browser details YourSeq 280 1358 1781 3000 85.9% chr7 + 99860137 99860593 457 browser details YourSeq 279 1374 2033 3000 80.6% chr14 + 16180055 16180564 510 browser details YourSeq 278 1401 2034 3000 83.3% chr8 - 85474822 85475286 465 browser details YourSeq 276 1343 2034 3000 80.6% chrX + 121039515 121040045 531 browser details YourSeq 276 1374 2034 3000 84.7% chr9 + 22268868 22269329 462 browser details YourSeq 275 1382 1770 3000 87.4% chr11 + 17917444 17917827 384 browser details YourSeq 274 1359 1795 3000 88.8% chr7 + 127157465 127157927 463 browser details YourSeq 268 1374 2035 3000 85.9% chr6 - 35578712 35579182 471 Note: The 3000 bp section upstream of Exon 1 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% chr2 + 152106199 152109198 3000 browser details YourSeq 186 2093 2286 3000 98.0% chr8 - 85422708 85422901 194 browser details YourSeq 186 2093 2287 3000 98.0% chr13 - 107630091 107630286 196 browser details YourSeq 186 2096 2292 3000 97.5% chrX + 108597609 108597806 198 browser details YourSeq 186 2092 2284 3000 98.5% chr11 + 97371235 97371428 194 browser details YourSeq 185 2092 2284 3000 98.0% chr8 + 107154354 107154546 193 browser details YourSeq 185 2093 2285 3000 98.0% chr8 + 84996607 84996799 193 browser details YourSeq 185 2093 2286 3000 98.0% chr3 + 95090734 95090928 195 browser details YourSeq 184 2097 2285 3000 99.0% chr6 - 97357549 97357842 294 browser details YourSeq 184 2095 2284 3000 97.4% chr14 - 52094976 52095164 189 browser details YourSeq 183 2095 2285 3000 98.0% chr9 + 44771146 44771336 191 browser details YourSeq 183 2093 2286 3000 95.9% chr4 + 123710592 123710783 192 browser details YourSeq 183 2092 2286 3000 97.0% chr4 + 123485081 123485275 195 browser details YourSeq 182 2090 2282 3000 97.5% chr5 - 137557297 137557490 194 browser details YourSeq 182 2091 2301 3000 92.7% chr15 - 99663606 99663812 207 browser details YourSeq 182 2092 2284 3000 97.5% chr11 - 103888394 103888588 195 browser details YourSeq 182 2093 2286 3000 97.0% chr16 + 17538437 17538630 194 browser details YourSeq 182 2095 2284 3000 96.9% chr11 + 99033832 99034020 189 browser details YourSeq 182 2095 2286 3000 97.4% chr10 + 76453786 76453977 192 browser details YourSeq 181 2092 2285 3000 94.8% chr8 + 105301719 105301908 190 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Srxn1 sulfiredoxin 1 homolog (S. cerevisiae) [ Mus musculus (house mouse) ] Gene ID: 76650, updated on 12-Aug-2019 Gene summary Official Symbol Srxn1 provided by MGI Official Full Name sulfiredoxin 1 homolog (S. cerevisiae) provided by MGI Primary source MGI:MGI:104971 See related Ensembl:ENSMUSG00000032802 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 Srx; Npn3; Srx1; TX01; AI854065; AW488194; 1700127B04Rik Expression Broad expression in duodenum adult (RPKM 100.0), adrenal adult (RPKM 95.3) and 21 other tissues See more Orthologs human all Genomic context Location: 2; 2 G3 See Srxn1 in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (152105524..152111376) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (151931466..151937089) Chromosome 2 - NC_000068.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Srxn1 ENSMUSG00000032802 Description sulfiredoxin 1 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:104971] Gene Synonyms 1700127B04Rik, Npn3, Srx, Srx1 Location Chromosome 2: 152,105,516-152,111,376 forward strand. GRCm38:CM000995.2 About this gene This gene has 3 transcripts (splice variants), 174 orthologues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Srxn1-201 ENSMUST00000041500.7 2812 155aa ENSMUSP00000046196.7 Protein coding CCDS16877 A2AQU8 TSL:1 GENCODE basic APPRIS P1 Srxn1-203 ENSMUST00000137751.1 446 No protein - lncRNA - - TSL:2 Srxn1-202 ENSMUST00000128882.1 375 No protein - lncRNA - - TSL:3 25.86 kb Forward strand 152.10Mb 152.11Mb 152.12Mb Genes (Comprehensive set... Scrt2-201 >protein coding Srxn1-202 >lncRNA Srxn1-203 >lncRNA Srxn1-201 >protein coding Contigs AL845161.5 > Regulatory Build 152.10Mb 152.11Mb 152.12Mb Reverse strand 25.86 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000041500 5.85 kb Forward strand Srxn1-201 >protein coding ENSMUSP00000046... Low complexity (Seg) Superfamily ParB/Sulfiredoxin superfamily SMART ParB/Sulfiredoxin Pfam ParB/Sulfiredoxin PIRSF Sulfiredoxin PANTHER Sulfiredoxin PTHR21348:SF2 Gene3D 3.90.1530.10 CDD cd16395 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 20 40 60 80 100 120 155 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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