https://www.alphaknockout.com Mouse Slc22a2 Knockout Project (CRISPR/Cas9) Objective: To create a Slc22a2 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Slc22a2 gene (NCBI Reference Sequence: NM_013667 ; Ensembl: ENSMUSG00000040966 ) is located on Mouse chromosome 17. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 11 (Transcript: ENSMUST00000046959). Exon 2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knockout allele are viable and fertile and display no obvious phenotypic abnormalities. No significant defects in the renal secretion of a model organic cation are observed. Exon 2 starts from about 25.02% of the coding region. Exon 2 covers 6.27% of the coding region. The size of effective KO region: ~104 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 11 Legends Exon of mouse Slc22a2 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(28.75% 575) | C(24.5% 490) | T(23.8% 476) | G(22.95% 459) Note: The 2000 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.95% 499) | C(25.8% 516) | T(29.95% 599) | G(19.3% 386) Note: The 2000 bp section downstream of Exon 2 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr17 + 12584779 12586778 2000 browser details YourSeq 63 875 947 2000 95.4% chr8 - 61736411 61736482 72 browser details YourSeq 45 427 566 2000 70.5% chr17 + 31603479 31603588 110 browser details YourSeq 43 992 1053 2000 97.9% chr3 - 112220238 112220305 68 browser details YourSeq 39 468 551 2000 74.5% chr1 + 128647344 128647411 68 browser details YourSeq 38 434 573 2000 68.0% chr6 - 114959495 114959611 117 browser details YourSeq 37 1017 1169 2000 63.5% chr5 + 131292076 131292149 74 browser details YourSeq 35 434 490 2000 80.8% chr14 + 61577010 61577066 57 browser details YourSeq 33 539 761 2000 51.3% chr13 + 37731300 37731347 48 browser details YourSeq 32 669 739 2000 97.1% chr13 + 116996867 116996938 72 browser details YourSeq 31 434 475 2000 84.7% chr11 - 29517736 29517776 41 browser details YourSeq 31 669 777 2000 94.3% chr1 + 154595845 154595954 110 browser details YourSeq 30 437 475 2000 89.5% chr13 - 17912065 17912104 40 browser details YourSeq 30 1017 1053 2000 78.8% chr13 - 6249484 6249516 33 browser details YourSeq 30 1014 1049 2000 91.7% chr6 + 45403845 45403880 36 browser details YourSeq 29 528 566 2000 87.2% chr16 + 13174558 13174596 39 browser details YourSeq 28 429 464 2000 88.9% chr6 - 29946486 29946521 36 browser details YourSeq 28 766 799 2000 91.2% chr2 - 147208629 147208662 34 browser details YourSeq 28 433 485 2000 90.0% chr2 - 117734266 117734317 52 browser details YourSeq 28 1020 1055 2000 93.8% chr1 - 40880033 40880068 36 Note: The 2000 bp section upstream of Exon 2 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 2000 1 2000 2000 100.0% chr17 + 12586883 12588882 2000 browser details YourSeq 67 1014 1245 2000 77.7% chr2 + 117256869 117257068 200 browser details YourSeq 52 1763 1814 2000 100.0% chr10 + 97719464 97719515 52 browser details YourSeq 51 965 1044 2000 89.3% chr5 - 19249738 19249821 84 browser details YourSeq 51 994 1226 2000 79.8% chr16 - 24155925 24156157 233 browser details YourSeq 46 1006 1108 2000 89.7% chr12 - 92118221 92118324 104 browser details YourSeq 45 979 1219 2000 85.0% chr11 + 92028848 92029086 239 browser details YourSeq 44 1005 1110 2000 67.0% chr11 - 9913422 9913524 103 browser details YourSeq 44 1065 1154 2000 86.7% chr10 + 70818332 70818435 104 browser details YourSeq 41 967 1203 2000 75.6% chr1 - 129038404 129038626 223 browser details YourSeq 36 1016 1079 2000 89.2% chr10 + 73185964 73186028 65 browser details YourSeq 36 969 1024 2000 78.2% chr1 + 26721380 26721434 55 browser details YourSeq 35 981 1030 2000 94.9% chr2 - 83151754 83151804 51 browser details YourSeq 35 1005 1045 2000 92.7% chrX + 167579374 167579414 41 browser details YourSeq 34 994 1047 2000 87.0% chrX + 71237457 71237511 55 browser details YourSeq 31 1191 1226 2000 94.3% chr4 + 22338639 22338674 36 browser details YourSeq 31 979 1029 2000 80.4% chr10 + 114847816 114847866 51 browser details YourSeq 30 1009 1044 2000 91.7% chr12 - 9401426 9401461 36 browser details YourSeq 30 989 1033 2000 94.2% chr1 - 195285952 195285997 46 browser details YourSeq 30 1185 1226 2000 81.1% chr5 + 3837933 3837972 40 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Slc22a2 solute carrier family 22 (organic cation transporter), member 2 [ Mus musculus (house mouse) ] Gene ID: 20518, updated on 24-Oct-2019 Gene summary Official Symbol Slc22a2 provided by MGI Official Full Name solute carrier family 22 (organic cation transporter), member 2 provided by MGI Primary source MGI:MGI:1335072 See related Ensembl:ENSMUSG00000040966 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 Oct2; Orct2 Expression Biased expression in kidney adult (RPKM 64.5), liver adult (RPKM 7.4) and 1 other tissueS ee more Orthologs human all Genomic context Location: 17 A1; 17 8.61 cM See Slc22a2 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (12584189..12628489) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (12777055..12821354) Chromosome 17 - NC_000083.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Slc22a2 ENSMUSG00000040966 Description solute carrier family 22 (organic cation transporter), member 2 [Source:MGI Symbol;Acc:MGI:1335072] Gene Synonyms Oct2, Orct2 Location Chromosome 17: 12,584,132-12,628,488 forward strand. GRCm38:CM001010.2 About this gene This gene has 2 transcripts (splice variants), 319 orthologues, 26 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Slc22a2-201 ENSMUST00000046959.8 2195 553aa ENSMUSP00000041186.7 Protein coding CCDS28392 O70577 TSL:1 GENCODE basic APPRIS P1 Slc22a2-202 ENSMUST00000233066.1 1977 544aa ENSMUSP00000156710.1 Protein coding - O70577 GENCODE basic 64.36 kb Forward strand 12.58Mb 12.59Mb 12.60Mb 12.61Mb 12.62Mb 12.63Mb Genes (Comprehensive set... Slc22a2-201 >protein coding Slc22a2-202 >protein coding Contigs AC167817.4 > Regulatory Build 12.58Mb 12.59Mb 12.60Mb 12.61Mb 12.62Mb 12.63Mb Reverse strand 64.36 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000046959 44.36 kb Forward strand Slc22a2-201 >protein coding ENSMUSP00000041... Transmembrane heli... Low complexity (Seg) TIGRFAM Organic cation transport protein/SVOP Superfamily MFS transporter superfamily Pfam Major facilitator, sugar transporter-like PROSITE profiles Major facilitator superfamily domain PROSITE patterns Sugar transporter, conserved site PANTHER PTHR24064:SF432 PTHR24064 Gene3D 1.20.1250.20 CDD cd17379 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 553 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.