https://www.alphaknockout.com Mouse Rbm7 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Rbm7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rbm7 gene (NCBI Reference Sequence: NM_144948 ; Ensembl: ENSMUSG00000042396 ) is located on Mouse chromosome 9. 5 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 5 (Transcript: ENSMUST00000170000). 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 Rbm7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-147I23 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 4 starts from about 43.77% 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: 2468 bp, and the size of intron 4 for 3'-loxP site insertion: 859 bp. The size of effective cKO region: ~627 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Rbm7 Homology arm 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. 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(7091bp) | A(28.15% 1996) | C(18.91% 1341) | T(32.68% 2317) | G(20.27% 1437) 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 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% chr9 - 48491189 48494188 3000 browser details YourSeq 207 5 782 3000 83.1% chr8 + 66845893 66846146 254 browser details YourSeq 203 29 779 3000 87.0% chr11 - 71057624 71057850 227 browser details YourSeq 146 1447 1636 3000 91.2% chr1 - 33749976 33750171 196 browser details YourSeq 143 1150 1619 3000 84.2% chr10 - 80378003 80378234 232 browser details YourSeq 143 1471 1634 3000 94.5% chr1 + 99902566 99902740 175 browser details YourSeq 139 1469 1635 3000 92.9% chrX + 7838638 7838996 359 browser details YourSeq 138 1470 1633 3000 93.8% chr8 + 105705885 105706056 172 browser details YourSeq 136 1474 1633 3000 93.7% chr14 - 25943011 25943434 424 browser details YourSeq 136 1468 1648 3000 88.8% chr10 + 71324501 71324686 186 browser details YourSeq 135 1468 1636 3000 92.1% chr4 - 135741535 135742101 567 browser details YourSeq 135 1468 1623 3000 93.6% chr19 + 46010631 46010791 161 browser details YourSeq 134 1468 1633 3000 91.9% chr11 - 120589888 120590056 169 browser details YourSeq 134 1468 1630 3000 94.1% chr4 + 46026362 46026528 167 browser details YourSeq 134 1468 1620 3000 94.8% chr19 + 3696621 3696781 161 browser details YourSeq 133 1468 1627 3000 92.5% chr8 - 34644247 34644411 165 browser details YourSeq 133 1468 1627 3000 93.6% chr17 - 68025481 68025646 166 browser details YourSeq 132 1468 1624 3000 94.2% chr11 - 79210571 79210818 248 browser details YourSeq 132 1474 1640 3000 95.4% chr5 + 113108877 113109052 176 browser details YourSeq 132 1468 1634 3000 90.4% chr1 + 21298492 21298663 172 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% chr9 - 48487598 48490597 3000 browser details YourSeq 1092 608 1900 3000 93.6% chr8 + 66846240 66847510 1271 browser details YourSeq 690 621 1409 3000 93.9% chr11 - 71056732 71057514 783 browser details YourSeq 132 2192 2368 3000 90.8% chr8 + 111011782 111011980 199 browser details YourSeq 130 2188 2348 3000 92.3% chr12 + 4168536 4168701 166 browser details YourSeq 128 2194 2348 3000 92.3% chr10 + 125819252 125819413 162 browser details YourSeq 127 2193 2340 3000 94.0% chrX - 77984532 77984686 155 browser details YourSeq 127 2183 2340 3000 92.1% chr5 - 124576259 124576426 168 browser details YourSeq 126 2204 2358 3000 91.7% chr9 + 78150009 78150179 171 browser details YourSeq 124 2181 2340 3000 88.1% chr15 - 59101647 59101794 148 browser details YourSeq 124 2193 2340 3000 92.6% chr10 - 85922690 85922838 149 browser details YourSeq 123 2190 2348 3000 85.9% chr6 + 27220832 27220986 155 browser details YourSeq 123 2197 2340 3000 95.0% chr14 + 98390179 98390337 159 browser details YourSeq 121 2204 2343 3000 93.6% chr10 + 125477196 125477337 142 browser details YourSeq 119 2193 2340 3000 91.1% chr12 - 88563425 88563596 172 browser details YourSeq 118 2199 2340 3000 92.2% chr8 + 29473954 29474109 156 browser details YourSeq 117 2204 2358 3000 92.1% chr12 + 52244687 52244841 155 browser details YourSeq 115 2174 2336 3000 90.9% chr5 + 24727701 24727986 286 browser details YourSeq 114 2181 2340 3000 90.3% chr2 - 97111686 97111855 170 browser details YourSeq 114 2204 2389 3000 84.5% chr1 - 50302171 50302346 176 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Rbm7 RNA binding motif protein 7 [ Mus musculus (house mouse) ] Gene ID: 67010, updated on 10-Oct-2019 Gene summary Official Symbol Rbm7 provided by MGI Official Full Name RNA binding motif protein 7 provided by MGI Primary source MGI:MGI:1914260 See related Ensembl:ENSMUSG00000042396 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 AU041934; AW554393; 1200007M24Rik; 1500011D06Rik Expression Ubiquitous expression in testis adult (RPKM 26.0), bladder adult (RPKM 18.9) and 28 other tissues See more Orthologs human all Genomic context Location: 9; 9 A5.3 See Rbm7 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (48488697..48495330, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (48296814..48303410, complement) Chromosome 9 - NC_000075.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Rbm7 ENSMUSG00000042396 Description RNA binding motif protein 7 [Source:MGI Symbol;Acc:MGI:1914260] Gene Synonyms 1200007M24Rik, 1500011D06Rik Location Chromosome 9: 48,488,701-48,495,299 reverse strand. GRCm38:CM001002.2 About this gene This gene has 4 transcripts (splice variants), 222 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rbm7- ENSMUST00000170000.3 1773 265aa ENSMUSP00000126374.2 Protein coding CCDS23155 Q14A95 TSL:1 201 Q9CQT2 GENCODE basic APPRIS P1 Rbm7- ENSMUST00000213276.1 2629 158aa ENSMUSP00000149397.1 Protein coding - A0A1L1SRC3 TSL:2 202 GENCODE basic Rbm7- ENSMUST00000214923.1 1867 39aa ENSMUSP00000150767.1 Nonsense mediated - A0A1L1SUJ3 TSL:1 204 decay Rbm7- ENSMUST00000213525.1 2703 No - Retained intron - - TSL:NA 203 protein 26.60 kb Forward strand 48.48Mb 48.49Mb 48.50Mb Genes Gm5617-201 >protein coding (Comprehensive set... Contigs AC160992.2 > Genes (Comprehensive set... < Rexo2-201protein coding < Rbm7-201protein coding < Rexo2-204protein coding < Rbm7-202protein coding < Rexo2-202retained intron < Rbm7-204nonsense mediated decay < Rexo2-210protein coding < Rbm7-203retained intron < Rexo2-209retained intron < Rexo2-208protein coding < Rexo2-205retained intron Regulatory Build 48.48Mb 48.49Mb 48.50Mb Reverse strand 26.60 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000170000 < Rbm7-201protein coding Reverse strand 6.60 kb ENSMUSP00000126... MobiDB lite Superfamily RNA-binding domain superfamily SMART RNA recognition motif domain Pfam RNA recognition motif domain PROSITE profiles RNA recognition motif domain PANTHER PTHR13798:SF4 PTHR13798 Gene3D Nucleotide-binding alpha-beta plait domain superfamily CDD RBM7, RNA recognition motif All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 265 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.