https://www.alphaknockout.com Mouse Ndufaf6 Knockout Project (CRISPR/Cas9) Objective: To create a Ndufaf6 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ndufaf6 gene (NCBI Reference Sequence: NM_001085493 ; Ensembl: ENSMUSG00000050323 ) is located on Mouse chromosome 4. 9 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000058183). Exon 2~4 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 19.82% of the coding region. Exon 2~4 covers 28.03% of the coding region. The size of effective KO region: ~6593 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 9 Legends Exon of mouse Ndufaf6 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 4 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(21.85% 437) | C(20.1% 402) | T(36.2% 724) | G(21.85% 437) 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(23.85% 477) | C(22.45% 449) | T(34.45% 689) | G(19.25% 385) Note: The 2000 bp section downstream of Exon 4 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% chr4 - 11073244 11075243 2000 browser details YourSeq 163 1095 1284 2000 93.2% chr9 + 73114351 73114541 191 browser details YourSeq 160 1095 1291 2000 94.0% chr10 + 79156641 79156838 198 browser details YourSeq 159 1098 1621 2000 85.2% chr13 + 57280375 57280837 463 browser details YourSeq 157 1106 1380 2000 86.1% chr11 + 121360081 121360295 215 browser details YourSeq 157 1134 1650 2000 88.7% chr11 + 78253982 78254489 508 browser details YourSeq 156 1095 1297 2000 92.9% chr14 + 66583164 66583627 464 browser details YourSeq 154 1095 1284 2000 93.3% chr7 + 68267959 68268148 190 browser details YourSeq 152 1106 1390 2000 84.4% chr19 + 41893029 41893226 198 browser details YourSeq 149 1094 1284 2000 87.8% chr11 + 121526810 121526990 181 browser details YourSeq 148 1095 1283 2000 93.7% chr19 - 43372328 43372528 201 browser details YourSeq 147 1094 1291 2000 88.8% chr2 - 16522595 16522779 185 browser details YourSeq 146 1098 1283 2000 88.9% chr2 - 73766990 73767166 177 browser details YourSeq 146 1095 1293 2000 87.5% chr16 - 9854825 9855013 189 browser details YourSeq 146 1096 1283 2000 91.2% chr15 + 76805489 76805674 186 browser details YourSeq 145 1094 1283 2000 88.4% chr19 - 5977049 5977226 178 browser details YourSeq 145 1095 1289 2000 84.9% chr3 + 33313522 33313699 178 browser details YourSeq 144 736 1283 2000 82.7% chr1 - 182926394 182926699 306 browser details YourSeq 143 1136 1393 2000 83.6% chr11 - 86955466 86955635 170 browser details YourSeq 143 1096 1294 2000 90.9% chr2 + 30058971 30059169 199 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% chr4 - 11064651 11066650 2000 browser details YourSeq 305 982 1296 2000 98.5% chr4 - 11065431 11065745 315 browser details YourSeq 305 904 1218 2000 98.5% chr4 - 11065357 11065671 315 browser details YourSeq 252 339 703 2000 93.8% chr1 + 59627191 59673014 45824 browser details YourSeq 185 337 687 2000 89.0% chr13 + 96544638 96545073 436 browser details YourSeq 180 339 722 2000 84.0% chr1 + 178456438 178456764 327 browser details YourSeq 173 350 684 2000 89.2% chr15 - 103078218 103078558 341 browser details YourSeq 168 350 680 2000 90.4% chr11 + 107518142 107518479 338 browser details YourSeq 162 346 679 2000 90.1% chr1 - 133009550 133010001 452 browser details YourSeq 159 339 541 2000 93.8% chr18 - 56522803 56523004 202 browser details YourSeq 157 339 541 2000 88.2% chr9 + 72310839 72311018 180 browser details YourSeq 157 339 547 2000 88.6% chr17 + 23284958 23285151 194 browser details YourSeq 156 323 540 2000 88.1% chr5 - 129935054 129935247 194 browser details YourSeq 153 323 540 2000 85.8% chr10 + 117098126 117098313 188 browser details YourSeq 147 340 550 2000 88.4% chrX + 113173862 113174060 199 browser details YourSeq 146 323 541 2000 85.5% chr15 + 55157194 55157388 195 browser details YourSeq 144 323 540 2000 85.0% chr9 + 66538581 66538779 199 browser details YourSeq 143 339 539 2000 87.6% chr11 + 15023807 15023989 183 browser details YourSeq 140 333 540 2000 83.6% chr10 - 128540767 128540953 187 browser details YourSeq 139 340 541 2000 87.2% chr18 + 32880916 32881099 184 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Ndufaf6 NADH:ubiquinone oxidoreductase complex assembly factor 6 [ Mus musculus (house mouse) ] Gene ID: 76947, updated on 12-Aug-2019 Gene summary Official Symbol Ndufaf6 provided by MGI Official Full Name NADH:ubiquinone oxidoreductase complex assembly factor 6 provided by MGI Primary source MGI:MGI:1924197 See related Ensembl:ENSMUSG00000050323 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 2310030N02Rik Expression Ubiquitous expression in adrenal adult (RPKM 4.2), kidney adult (RPKM 4.2) and 28 other tissues See more Orthologs human all Genomic context Location: 4; 4 A1 See Ndufaf6 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (11048991..11076237, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (10978192..11003351, complement) Chromosome 4 - NC_000070.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Ndufaf6 ENSMUSG00000050323 Description NADH:ubiquinone oxidoreductase complex assembly factor 6 [Source:MGI Symbol;Acc:MGI:1924197] Gene Synonyms 2310030N02Rik Location Chromosome 4: 11,051,045-11,076,205 reverse strand. GRCm38:CM000997.2 About this gene This gene has 7 transcripts (splice variants), 191 orthologues, 2 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 Ndufaf6-201 ENSMUST00000058183.8 1082 333aa ENSMUSP00000062039.8 Protein coding CCDS38691 A2AIL4 TSL:1 GENCODE basic APPRIS P1 Ndufaf6-203 ENSMUST00000126468.7 863 No protein - lncRNA - - TSL:5 Ndufaf6-202 ENSMUST00000124607.7 675 No protein - lncRNA - - TSL:5 Ndufaf6-207 ENSMUST00000154122.1 528 No protein - lncRNA - - TSL:5 Ndufaf6-204 ENSMUST00000141019.1 461 No protein - lncRNA - - TSL:1 Ndufaf6-206 ENSMUST00000148127.1 352 No protein - lncRNA - - TSL:5 Ndufaf6-205 ENSMUST00000144475.1 248 No protein - lncRNA - - TSL:3 45.16 kb Forward strand 11.05Mb 11.06Mb 11.07Mb 11.08Mb Contigs AL732497.15 > Genes (Comprehensive set... < Ndufaf6-201protein coding < Ndufaf6-205lncRNA < Ndufaf6-207lncRNA < Ndufaf6-203lncRNA < Ndufaf6-202lncRNA < Ndufaf6-206lncRNA < Ndufaf6-204lncRNA Regulatory Build 11.05Mb 11.06Mb 11.07Mb 11.08Mb Reverse strand 45.16 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000058183 < Ndufaf6-201protein coding Reverse strand 25.16 kb ENSMUSP00000062... Low complexity (Seg) Superfamily Isoprenoid synthase domain superfamily Pfam PF00494 PANTHER PTHR21181 PTHR21181:SF13 Gene3D Isoprenoid synthase domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend frameshift variant missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 333 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.