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Mouse Rsf1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Rsf1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rsf1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Rsf1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rsf1 gene (NCBI Reference Sequence: NM_001081267 ; Ensembl: ENSMUSG00000035623 ) is located on Mouse chromosome 7. 16 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 16 (Transcript: ENSMUST00000107153). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rsf1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-66B7 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 conditional allele activated in neurons exhibit increased susceptibility to etoposide-induced neuron apoptosis with failure to repair double-strand breaks. Exon 4 starts from about 8.98% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2835 bp, and the size of intron 4 for 3'-loxP site insertion: 13542 bp. The size of effective cKO region: ~706 bp. The cKO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 16 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Rsf1 Homology arm cKO region loxP site Page 2 of 9 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(7206bp) | A(28.85% 2079) | C(17.6% 1268) | T(34.21% 2465) | G(19.34% 1394) 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 9 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% chr7 + 97636056 97639055 3000 browser details YourSeq 122 1616 1799 3000 86.4% chrX + 101279839 101280181 343 browser details YourSeq 119 1614 1823 3000 85.0% chr1 + 25005385 25005599 215 browser details YourSeq 110 1624 1810 3000 84.7% chr19 - 15947620 15947960 341 browser details YourSeq 107 1621 1810 3000 84.6% chr5 - 68786365 68786711 347 browser details YourSeq 107 1665 1810 3000 84.8% chr18 - 33904234 33904377 144 browser details YourSeq 105 1629 1816 3000 87.8% chr10 - 119178807 119178985 179 browser details YourSeq 102 1661 1810 3000 84.7% chr9 + 53711464 53711616 153 browser details YourSeq 101 1664 1811 3000 81.4% chrX + 163449141 163449285 145 browser details YourSeq 99 1648 1808 3000 91.1% chr10 - 70057744 70057914 171 browser details YourSeq 99 1665 1811 3000 84.3% chr1 - 151504114 151504284 171 browser details YourSeq 97 1608 1811 3000 77.1% chr14 - 98085828 98085982 155 browser details YourSeq 97 1664 1815 3000 80.0% chr10 - 100555131 100555280 150 browser details YourSeq 97 1646 1812 3000 78.5% chr1 - 148660161 148660323 163 browser details YourSeq 97 1648 1800 3000 79.2% chr18 + 75729575 75729721 147 browser details YourSeq 95 1663 1806 3000 83.4% chr8 + 83361966 83362128 163 browser details YourSeq 95 1664 1810 3000 83.9% chr15 + 59964955 59965142 188 browser details YourSeq 95 1664 1797 3000 85.9% chr13 + 7639458 7639643 186 browser details YourSeq 93 1644 1796 3000 86.1% chr11 - 87958344 87958508 165 browser details YourSeq 92 1618 1810 3000 75.8% chr11 + 106484759 106484891 133 Note: The 3000 bp section upstream of Exon 4 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% chr7 + 97639762 97642761 3000 browser details YourSeq 79 536 655 3000 86.7% chr1 - 25040212 25040333 122 browser details YourSeq 72 537 655 3000 81.8% chr14 + 34318368 34318506 139 browser details YourSeq 67 562 652 3000 89.6% chr6 - 82833724 82833836 113 browser details YourSeq 66 547 651 3000 87.5% chr2 - 91076110 91076307 198 browser details YourSeq 64 539 655 3000 97.1% chr1 - 156536513 156536643 131 browser details YourSeq 64 1213 1588 3000 68.7% chr11 + 51633320 51633491 172 browser details YourSeq 63 561 646 3000 88.9% chr6 - 134607990 134608093 104 browser details YourSeq 62 525 627 3000 81.1% chr5 - 28283001 28283097 97 browser details YourSeq 62 571 658 3000 88.8% chr3 - 127206803 127206892 90 browser details YourSeq 62 573 655 3000 89.8% chr11 - 5317343 5317427 85 browser details YourSeq 62 571 655 3000 88.7% chr10 - 63277989 63278074 86 browser details YourSeq 61 580 655 3000 93.0% chr12 + 78486689 78486766 78 browser details YourSeq 60 573 654 3000 90.6% chr4 - 154615367 154615448 82 browser details YourSeq 59 571 655 3000 90.5% chr5 + 144054760 144054845 86 browser details YourSeq 58 1123 1335 3000 76.4% chr8 - 114782732 114782927 196 browser details YourSeq 58 575 655 3000 88.2% chr7 + 101219976 101220058 83 browser details YourSeq 57 573 655 3000 86.9% chr2 - 73022801 73022883 83 browser details YourSeq 57 573 655 3000 84.4% chr17 + 83844254 83844336 83 browser details YourSeq 56 555 655 3000 82.6% chr2 - 87541308 87541428 121 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 9 https://www.alphaknockout.com Gene and protein information: Rsf1 remodeling and spacing factor 1 [ Mus musculus (house mouse) ] Gene ID: 233532, updated on 12-Aug-2019 Gene summary Official Symbol Rsf1 provided by MGI Official Full Name remodeling and spacing factor 1 provided by MGI Primary source MGI:MGI:2682305 See related Ensembl:ENSMUSG00000035623 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 XAP8; p325; Gm164; Hbxap; 4832420A03Rik; C030033M12Rik Expression Broad expression in CNS E11.5 (RPKM 4.7), CNS E14 (RPKM 4.1) and 24 other tissues See more Orthologs human all Genomic context Location: 7; 7 E1 See Rsf1 in Genome Data Viewer Exon count: 17 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (97579870..97692782) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (104728406..104841292) Chromosome 7 - NC_000073.6 Page 5 of 9 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Rsf1 ENSMUSG00000035623 Description remodeling and spacing factor 1 [Source:MGI Symbol;Acc:MGI:2682305] Gene Synonyms 4832420A03Rik, C030033M12Rik, Hbxap, XAP8, p325 Location Chromosome 7: 97,579,889-97,692,778 forward strand. GRCm38:CM001000.2 About this gene This gene has 8 transcripts (splice variants), 233 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rsf1- ENSMUST00000107153.2 11124 1441aa ENSMUSP00000102771.2 Protein coding CCDS40025 E9PWW9 TSL:5 201 GENCODE basic APPRIS P1 Rsf1- ENSMUST00000123731.7 2493 14aa ENSMUSP00000145663.1 Nonsense mediated - A0A0U1RNQ5 CDS 5' 202 decay incomplete TSL:5 Rsf1- ENSMUST00000135270.2 764 121aa ENSMUSP00000145808.1 Nonsense mediated - A0A0U1RP31 CDS 5' 204 decay incomplete TSL:5 Rsf1- ENSMUST00000127891.2 754 109aa ENSMUSP00000145905.1 Nonsense mediated - A0A0U1RPA7 CDS 5' 203 decay incomplete TSL:3 Rsf1- ENSMUST00000156060.7 5695 No - Retained intron - - TSL:2 206 protein Rsf1- ENSMUST00000206866.1 3635 No - Retained intron - - TSL:NA 208 protein Rsf1- ENSMUST00000205536.1 3085 No - Retained intron - - TSL:NA 207 protein Rsf1- ENSMUST00000150288.6 542 No - Retained intron - - TSL:5 205 protein Page 6 of 9 https://www.alphaknockout.com 132.89 kb Forward strand 97.60Mb 97.65Mb 97.70Mb Genes (Comprehensive set... Rsf1-207 >retained intron Rsf1-208 >retained intron Clns1a-202 >retained intron Rsf1-201 >protein coding Rsf1-206 >retained intron Rsf1-203 >nonsense mediated decay Rsf1-202 >nonsense mediated decay Rsf1-205 >retained intron Rsf1-204 >nonsense mediated decay Clns1a-201 >protein coding Contigs AC149605.7 > < AC124541.4 AC151477.1 > Genes < Aamdc-201protein coding < Ywhaq-ps1-201processed pseudogene < Rsf1os2-203lncRNA (Comprehensive set... < Aamdc-214protein coding < Rpl15-ps5-201processed pseudogene < Gm45186-201processed pseudogene < Rsf1os2-202lncRNA < Aamdc-206protein coding < Rsf1os1-201lncRNA < Rsf1os2-201lncRNA < Aamdc-202protein coding < Aamdc-213protein coding < Aamdc-204nonsense mediated decay < Aamdc-208lncRNA < Aamdc-203protein coding <
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