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Mouse Rbm22 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Rbm22 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rbm22 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Rbm22 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rbm22 gene (NCBI Reference Sequence: NM_025776 ; Ensembl: ENSMUSG00000024604 ) is located on Mouse chromosome 18. 11 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000025506). 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 Rbm22 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-119L17 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 11.03% 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: 567 bp, and the size of intron 4 for 3'-loxP site insertion: 1122 bp. The size of effective cKO region: ~633 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 3 4 5 6 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Rbm22 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(7133bp) | A(25.84% 1843) | C(22.33% 1593) | T(30.16% 2151) | G(21.67% 1546) 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% chr18 + 60561114 60564113 3000 browser details YourSeq 157 895 1229 3000 93.5% chr15 + 100286865 100287722 858 browser details YourSeq 151 906 2441 3000 90.4% chr10 - 128403791 128533551 129761 browser details YourSeq 148 880 1057 3000 93.0% chr11 - 115129157 115129346 190 browser details YourSeq 147 862 1057 3000 86.4% chr4 - 107408620 107408788 169 browser details YourSeq 147 881 1057 3000 92.5% chr4 + 23798097 23798275 179 browser details YourSeq 145 879 1058 3000 91.6% chr15 + 85556488 85556679 192 browser details YourSeq 144 891 1229 3000 84.9% chr14 - 47400572 47400880 309 browser details YourSeq 139 881 1058 3000 90.5% chr10 - 85639188 85639370 183 browser details YourSeq 137 891 1050 3000 91.3% chr8 - 93464174 93464323 150 browser details YourSeq 137 915 1227 3000 85.9% chr11 + 85299413 85299688 276 browser details YourSeq 137 884 1048 3000 89.3% chr1 + 68415761 68415920 160 browser details YourSeq 136 889 1057 3000 93.0% chr13 - 28938583 28938755 173 browser details YourSeq 134 895 1049 3000 91.1% chr14 - 56891863 56892009 147 browser details YourSeq 134 891 1059 3000 87.8% chr13 - 30098780 30098944 165 browser details YourSeq 134 886 1047 3000 93.0% chr6 + 34908510 34908672 163 browser details YourSeq 133 889 1049 3000 92.9% chr4 - 98770490 98770653 164 browser details YourSeq 133 890 1050 3000 93.3% chr13 - 67861043 67861202 160 browser details YourSeq 133 895 1049 3000 97.2% chr12 - 105746259 105746777 519 browser details YourSeq 133 890 1048 3000 93.6% chr1 - 146366323 146366494 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% chr18 + 60564747 60567746 3000 browser details YourSeq 134 2269 2449 3000 89.6% chr16 + 17476330 17476500 171 browser details YourSeq 133 2278 2452 3000 90.0% chr4 + 150105223 150105390 168 browser details YourSeq 132 2275 2451 3000 95.3% chr9 - 101067427 101067822 396 browser details YourSeq 132 2275 2449 3000 89.8% chr1 + 131919951 131920113 163 browser details YourSeq 131 2276 2449 3000 91.0% chr16 - 33220461 33220626 166 browser details YourSeq 131 2269 2442 3000 89.8% chrX + 90991201 90991361 161 browser details YourSeq 130 2278 2454 3000 95.8% chr3 - 90752035 90752353 319 browser details YourSeq 130 2273 2442 3000 89.7% chr16 - 17471339 17471498 160 browser details YourSeq 130 2273 2442 3000 89.1% chr10 - 108566266 108566423 158 browser details YourSeq 129 2275 2442 3000 90.8% chr9 - 35223838 35223992 155 browser details YourSeq 129 2273 2438 3000 91.3% chr4 - 135822939 135823088 150 browser details YourSeq 129 2273 2442 3000 90.7% chr17 - 65894823 65894979 157 browser details YourSeq 129 2273 2445 3000 90.3% chr1 + 54431382 54431546 165 browser details YourSeq 128 2275 2441 3000 91.2% chr4 - 148225144 148225297 154 browser details YourSeq 128 2273 2438 3000 90.6% chr4 - 74211344 74211493 150 browser details YourSeq 128 2275 2445 3000 89.6% chr10 - 45062564 45062721 158 browser details YourSeq 128 2273 2451 3000 93.4% chr2 + 31531603 31531922 320 browser details YourSeq 128 2273 2442 3000 90.6% chr2 + 24849059 24849215 157 browser details YourSeq 128 2273 2453 3000 94.5% chr17 + 32460574 32460817 244 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: Rbm22 RNA binding motif protein 22 [ Mus musculus (house mouse) ] Gene ID: 66810, updated on 10-Oct-2019 Gene summary Official Symbol Rbm22 provided by MGI Official Full Name RNA binding motif protein 22 provided by MGI Primary source MGI:MGI:1914060 See related Ensembl:ENSMUSG00000024604 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as 8430430L24Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 18.7), CNS E14 (RPKM 17.4) and 28 other tissues See more Orthologs human all Genomic context Location: 18; 18 D3 See Rbm22 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (60560786..60572729) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (60720440..60732383) Chromosome 18 - NC_000084.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Rbm22 ENSMUSG00000024604 Description RNA binding motif protein 22 [Source:MGI Symbol;Acc:MGI:1914060] Gene Synonyms 8430430L24Rik Location Chromosome 18: 60,560,736-60,572,810 forward strand. GRCm38:CM001011.2 About this gene This gene has 5 transcripts (splice variants), 227 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rbm22-201 ENSMUST00000025506.6 2210 420aa ENSMUSP00000025506.6 Protein coding CCDS29272 Q8BHS3 TSL:1 GENCODE basic APPRIS P1 Rbm22-205 ENSMUST00000161544.1 3541 No protein - Retained intron - - TSL:1 Rbm22-202 ENSMUST00000159203.1 595 No protein - Retained intron - - TSL:2 Rbm22-204 ENSMUST00000160353.1 573 No protein - Retained intron - - TSL:2 Rbm22-203 ENSMUST00000160020.1 352 No protein - Retained intron - - TSL:2 32.08 kb Forward strand 60.56Mb 60.57Mb 60.58Mb Genes (Comprehensive set... Dctn4-204 >protein coding Rbm22-201 >protein coding Dctn4-201 >protein coding Rbm22-203 >retained intron Rbm22-202 >retained intron Dctn4-203 >retained intron Rbm22-205 >retained intron Rbm22-204 >retained intron Dctn4-206 >retained intron Contigs < AC135638.5 < AC149216.5 Genes < Myoz3-201protein coding (Comprehensive set... Regulatory Build 60.56Mb 60.57Mb 60.58Mb Reverse strand 32.08 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000025506 12.07 kb Forward strand Rbm22-201 >protein coding ENSMUSP00000025... MobiDB lite Low complexity (Seg) Superfamily RNA-binding domain superfamily Zinc finger, CCCH-type superfamily SMART Zinc finger, CCCH-type RNA recognition motif domain Pfam RNA recognition motif domain PROSITE profiles RNA recognition motif domain Zinc finger, CCCH-type PANTHER Pre-mRNA-splicing factor Cwc2/Slt11 PTHR14089:SF7 Gene3D 4.10.1000.10 Nucleotide-binding alpha-beta plait domain superfamily CDD cd12224 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 420 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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