https://www.alphaknockout.com Mouse Mrps24 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mrps24 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mrps24 gene (NCBI Reference Sequence: NM_026080 ; Ensembl: ENSMUSG00000020477 ) is located on Mouse chromosome 11. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000154330). 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 Mrps24 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-387E5 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 covers 56.09% of the coding region. Start codon is in exon 1, and stop codon is in exon 4. The size of intron 3 for 5'-loxP site insertion: 2565 bp. The size of effective cKO region: ~1500 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 5' gRNA region gRNA region 3' 1 4 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mrps24 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. 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(6781bp) | A(24.48% 1660) | C(22.7% 1539) | T(27.62% 1873) | G(25.2% 1709) 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% chr11 - 5704983 5707982 3000 browser details YourSeq 160 2301 2579 3000 87.3% chr16 - 14093161 14093369 209 browser details YourSeq 160 2379 2580 3000 93.6% chr19 + 7248820 7249104 285 browser details YourSeq 159 2388 2579 3000 93.6% chr13 + 58367231 58367426 196 browser details YourSeq 157 2395 2579 3000 92.7% chr5 - 102731253 102731435 183 browser details YourSeq 157 2411 2581 3000 96.0% chr2 - 164511712 164511882 171 browser details YourSeq 157 2392 2571 3000 96.0% chr3 + 88963176 88963366 191 browser details YourSeq 153 2408 2579 3000 95.3% chr2 + 103905309 103905681 373 browser details YourSeq 150 2380 2573 3000 87.8% chr13 - 112861665 112861835 171 browser details YourSeq 145 2417 2579 3000 94.5% chr9 + 115830737 115830899 163 browser details YourSeq 142 2417 2579 3000 93.9% chr5 - 90203584 90203751 168 browser details YourSeq 136 2417 2579 3000 92.0% chrX + 101446819 101446981 163 browser details YourSeq 134 2439 2579 3000 97.9% chr15 - 73500057 73500323 267 browser details YourSeq 129 2417 2570 3000 93.3% chr18 + 11669704 11669865 162 browser details YourSeq 122 2431 2570 3000 93.6% chr12 - 110720852 110720991 140 browser details YourSeq 122 2445 2580 3000 94.9% chr11 + 84061078 84061213 136 browser details YourSeq 119 2416 2544 3000 96.2% chr9 - 113925906 113926034 129 browser details YourSeq 96 1 103 3000 99.0% chr7 + 82624741 82624846 106 browser details YourSeq 96 1 107 3000 91.0% chr2 + 122542403 122542501 99 browser details YourSeq 96 1 108 3000 97.1% chr12 + 87490032 87490141 110 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% chr11 - 5701452 5704451 3000 browser details YourSeq 643 1303 2212 3000 89.1% chr6 - 141916054 142141636 225583 browser details YourSeq 628 1341 2203 3000 90.8% chr6 + 103156720 103157632 913 browser details YourSeq 623 1412 2204 3000 91.6% chr19 - 55169258 55170112 855 browser details YourSeq 611 1397 2204 3000 90.2% chr3 - 103071634 103072509 876 browser details YourSeq 604 1312 2146 3000 88.9% chr1 - 41844901 41845779 879 browser details YourSeq 601 1370 2204 3000 88.8% chr16 + 66128936 66129849 914 browser details YourSeq 593 1383 2206 3000 88.9% chr8 - 72059737 72060622 886 browser details YourSeq 593 1428 2205 3000 90.5% chr6 - 10900926 10901749 824 browser details YourSeq 588 1364 2204 3000 89.1% chr9 + 119857357 119858246 890 browser details YourSeq 574 1368 2205 3000 89.6% chr6 + 41330755 41331670 916 browser details YourSeq 573 1367 2205 3000 89.8% chr12 + 111522142 111523054 913 browser details YourSeq 570 1383 2204 3000 89.0% chr19 - 17543981 17544863 883 browser details YourSeq 567 1382 2142 3000 90.0% chr6 - 11678596 11679412 817 browser details YourSeq 563 1318 2145 3000 86.9% chr1 + 100806468 100807347 880 browser details YourSeq 562 1412 2204 3000 90.4% chr3 - 21747304 21748177 874 browser details YourSeq 559 1303 2205 3000 90.9% chr13 - 13842728 13843660 933 browser details YourSeq 558 1410 2202 3000 90.9% chr19 - 30219459 30220494 1036 browser details YourSeq 554 1422 2204 3000 89.1% chr1 + 44328951 44329770 820 browser details YourSeq 552 1423 2192 3000 89.9% chr3 - 42523330 42524160 831 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: Mrps24 mitochondrial ribosomal protein S24 [ Mus musculus (house mouse) ] Gene ID: 64660, updated on 12-Aug-2019 Gene summary Official Symbol Mrps24 provided by MGI Official Full Name mitochondrial ribosomal protein S24 provided by MGI Primary source MGI:MGI:1928142 See related Ensembl:ENSMUSG00000020477 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 S24mt; Rpms24; MRP-S24; AI414579; 3110030K20Rik Expression Ubiquitous expression in adrenal adult (RPKM 188.3), stomach adult (RPKM 152.2) and 27 other tissues See more Orthologs human all Genomic context Location: 11; 11 A1 See Mrps24 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (5703982..5707701, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (5603986..5607702, complement) Chromosome 11 - NC_000077.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Mrps24 ENSMUSG00000020477 Description mitochondrial ribosomal protein S24 [Source:MGI Symbol;Acc:MGI:1928142] Gene Synonyms 3110030K20Rik, Rpms24 Location Chromosome 11: 5,703,983-5,715,680 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 200 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mrps24-205 ENSMUST00000154330.1 995 167aa ENSMUSP00000119535.1 Protein coding CCDS24402 Q9CQV5 TSL:1 GENCODE basic APPRIS P1 Mrps24-201 ENSMUST00000020770.10 700 No protein - Retained intron - - TSL:2 Mrps24-203 ENSMUST00000132874.1 503 No protein - Retained intron - - TSL:3 Mrps24-202 ENSMUST00000123697.1 418 No protein - Retained intron - - TSL:2 Mrps24-204 ENSMUST00000149980.1 714 No protein - lncRNA - - TSL:3 31.70 kb Forward strand 5.70Mb 5.71Mb 5.72Mb Contigs AL627069.10 > Genes (Comprehensive set... < Mrps24-205protein coding < Urgcp-201protein coding < Mrps24-201retained intron < Urgcp-206protein coding < Mrps24-204lncRNA < Urgcp-205protein coding < Mrps24-203retained intron < Urgcp-202protein coding < Mrps24-202retained intron < Urgcp-204protein coding < Urgcp-203protein coding Regulatory Build 5.70Mb 5.71Mb 5.72Mb Reverse strand 31.70 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000154330 < Mrps24-205protein coding Reverse strand 3.71 kb ENSMUSP00000119... Low complexity (Seg) Pfam 28S ribosomal protein S24, mitochondrial PANTHER 28S ribosomal protein S24, mitochondrial All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained frameshift variant missense variant splice region variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 167 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.