https://www.alphaknockout.com Mouse Slc25a16 Knockout Project (CRISPR/Cas9) Objective: To create a Slc25a16 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Slc25a16 gene (NCBI Reference Sequence: NM_175194 ; Ensembl: ENSMUSG00000071253 ) is located on Mouse chromosome 10. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000044977). Exon 2~5 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: Exon 2 starts from about 13.15% of the coding region. Exon 2~5 covers 41.47% of the coding region. The size of effective KO region: ~9244 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 3 4 5 9 Legends Exon of mouse Slc25a16 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 388 bp section downstream of Exon 5 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. 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(21.8% 436) | C(23.35% 467) | T(32.75% 655) | G(22.1% 442) 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(388bp) | A(27.84% 108) | C(18.56% 72) | T(26.55% 103) | G(27.06% 105) Note: The 388 bp section downstream of Exon 5 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% chr10 + 62926297 62928296 2000 browser details YourSeq 379 1 1057 2000 97.8% chr6 + 37883070 38558606 675537 browser details YourSeq 234 590 1057 2000 91.5% chr16 + 64892411 64892691 281 browser details YourSeq 218 1 619 2000 89.1% chr11 - 82963630 82963986 357 browser details YourSeq 208 1 630 2000 93.7% chr2 - 32143934 32144547 614 browser details YourSeq 208 1 349 2000 94.5% chr19 + 42065435 42066014 580 browser details YourSeq 206 1 344 2000 96.9% chr9 - 44958915 44959281 367 browser details YourSeq 206 1159 1721 2000 89.9% chr13 - 28803468 28803722 255 browser details YourSeq 202 855 1058 2000 99.6% chr9 - 35328218 35328421 204 browser details YourSeq 202 1 633 2000 89.0% chr13 - 58171699 58172149 451 browser details YourSeq 200 1 405 2000 97.2% chr7 - 49348876 49349373 498 browser details YourSeq 198 855 1056 2000 99.1% chr3 - 97085955 97086156 202 browser details YourSeq 197 22 621 2000 88.0% chr5 - 137712333 137712622 290 browser details YourSeq 196 863 1058 2000 100.0% chr17 - 35416674 35416869 196 browser details YourSeq 196 863 1058 2000 100.0% chr17 - 35461880 35462075 196 browser details YourSeq 196 1 334 2000 97.2% chr6 + 5556447 5556998 552 browser details YourSeq 194 1 532 2000 88.5% chr10 - 128432852 128433071 220 browser details YourSeq 193 860 1057 2000 97.5% chr1 + 156533782 156533977 196 browser details YourSeq 192 1 633 2000 88.3% chr2 - 120907228 120907531 304 browser details YourSeq 192 858 1056 2000 97.0% chr1 + 120060132 120060327 196 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 388 1 388 388 100.0% chr10 + 62937541 62937928 388 browser details YourSeq 55 174 233 388 96.7% chr10 + 71927026 71927088 63 browser details YourSeq 54 175 233 388 96.7% chr10 + 71926857 71926918 62 browser details YourSeq 53 175 233 388 96.6% chr4 - 58578368 58578474 107 browser details YourSeq 52 175 234 388 94.9% chr11 + 55368679 55368841 163 browser details YourSeq 51 175 233 388 89.7% chr2 - 20029448 20029505 58 browser details YourSeq 50 178 233 388 94.7% chr18 - 82119489 82119544 56 browser details YourSeq 50 180 233 388 96.3% chr12 - 116623277 116623330 54 browser details YourSeq 49 172 233 388 90.0% chr8 - 126524113 126524176 64 browser details YourSeq 49 175 233 388 93.0% chr19 - 56353070 56353143 74 browser details YourSeq 49 181 233 388 96.3% chr2 + 9904835 9904887 53 browser details YourSeq 49 175 232 388 87.1% chr10 + 25964944 25964998 55 browser details YourSeq 48 174 233 388 92.8% chr9 + 111923632 111923694 63 browser details YourSeq 47 175 233 388 88.5% chr19 + 4494255 4494311 57 browser details YourSeq 47 175 233 388 82.0% chr1 + 109660568 109660618 51 browser details YourSeq 45 175 233 388 78.5% chr8 - 32851203 32851253 51 browser details YourSeq 45 175 233 388 78.5% chr2 - 172051442 172051492 51 browser details YourSeq 45 175 233 388 78.5% chr17 - 45240946 45240996 51 browser details YourSeq 45 178 233 388 96.0% chr15 - 97945593 97945651 59 browser details YourSeq 45 175 233 388 78.5% chr13 - 98480699 98480749 51 Note: The 388 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Slc25a16 solute carrier family 25 (mitochondrial carrier, Graves disease autoantigen), member 16 [ Mus musculus (house mouse) ] Gene ID: 73132, updated on 12-Aug-2019 Gene summary Official Symbol Slc25a16 provided by MGI Official Full Name solute carrier family 25 (mitochondrial carrier, Graves disease autoantigen), member 16 provided by MGI Primary source MGI:MGI:1920382 See related Ensembl:ENSMUSG00000071253 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 GDA; GDC; ML7; HGT.1; 3110021G18Rik Expression Ubiquitous expression in kidney adult (RPKM 38.4), adrenal adult (RPKM 23.7) and 27 other tissues See more Orthologs human all Genomic context Location: 10; 10 B4 See Slc25a16 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (62920632..62946494) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (62383381..62409242) Chromosome 10 - NC_000076.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Slc25a16 ENSMUSG00000071253 Description solute carrier family 25 (mitochondrial carrier, Graves disease autoantigen), member 16 [Source:MGI Symbol;Acc:MGI:1920382] Gene Synonyms 3110021G18Rik, HGT.1, ML7 Location Chromosome 10: 62,920,633-62,946,498 forward strand. GRCm38:CM001003.2 About this gene This gene has 4 transcripts (splice variants), 191 orthologues, 48 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Slc25a16-201 ENSMUST00000044977.9 3145 332aa ENSMUSP00000043370.3 Protein coding CCDS23893 Q8C0K5 TSL:1 GENCODE basic APPRIS P1 Slc25a16-203 ENSMUST00000144459.1 1038 322aa ENSMUSP00000114510.1 Protein coding - D3YVC9 CDS 3' incomplete TSL:5 Slc25a16-202 ENSMUST00000138597.1 1744 No protein - Retained intron - - TSL:1 Slc25a16-204 ENSMUST00000152350.1 398 No protein - Retained intron - - TSL:2 45.87 kb Forward strand 62.92Mb 62.93Mb 62.94Mb 62.95Mb Genes Gm22803-201 >misc RNA Gm16128-201 >processed pseudogene Dna2-201 >nonsense mediated decay (Comprehensive set... Slc25a16-201 >protein coding Dna2-203 >protein coding Slc25a16-202 >retained intron Slc25a16-204 >retained intron Slc25a16-203 >protein coding Dna2-204 >retained intron Dna2-202 >retained intron Contigs < AC153942.8 AC113107.15 > Genes < Gm25862-201snoRNA (Comprehensive set... Regulatory Build 62.92Mb 62.93Mb 62.94Mb 62.95Mb Reverse strand 45.87 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript pseudogene RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000044977 25.87 kb Forward strand Slc25a16-201 >protein coding ENSMUSP00000043... Low complexity (Seg) Superfamily Mitochondrial carrier domain superfamily Prints Graves disease carrier protein Mitochondrial carrier protein Pfam Mitochondrial substrate/solute carrier PROSITE profiles Mitochondrial substrate/solute carrier PANTHER PTHR24089:SF0 PTHR24089 Gene3D Mitochondrial carrier domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 332 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.