https://www.alphaknockout.com Mouse Acss3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Acss3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Acss3 gene (NCBI Reference Sequence: NM_001142804 ; Ensembl: ENSMUSG00000035948 ) is located on Mouse chromosome 10. 16 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000165067). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Acss3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-414G20 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 2 starts from about 14.52% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 38336 bp, and the size of intron 2 for 3'-loxP site insertion: 31290 bp. The size of effective cKO region: ~645 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 2 16 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Acss3 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(7145bp) | A(25.08% 1792) | C(19.23% 1374) | T(36.29% 2593) | G(19.4% 1386) Note: The sequence of homologous arms and cKO region 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 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% chr10 - 107085202 107088201 3000 browser details YourSeq 226 530 846 3000 86.3% chr7 - 129275793 129276117 325 browser details YourSeq 225 535 846 3000 86.7% chr5 + 125527978 125528297 320 browser details YourSeq 221 208 843 3000 80.4% chr1 + 136538906 136539238 333 browser details YourSeq 217 538 846 3000 84.0% chr15 + 75668108 75668413 306 browser details YourSeq 212 572 846 3000 89.3% chr9 + 105583558 105583840 283 browser details YourSeq 210 530 846 3000 84.8% chr4 - 42295712 42296038 327 browser details YourSeq 210 530 846 3000 84.8% chr4 - 41943482 41943808 327 browser details YourSeq 210 570 855 3000 87.2% chr13 - 41464167 41464452 286 browser details YourSeq 210 530 846 3000 84.8% chr4_JH584293_random + 167520 167846 327 browser details YourSeq 210 530 846 3000 84.8% chr4_GL456350_random + 107809 108135 327 browser details YourSeq 208 530 846 3000 84.5% chr4 - 42198417 42198743 327 browser details YourSeq 208 398 839 3000 88.3% chr3 - 146480233 146480775 543 browser details YourSeq 208 534 846 3000 87.8% chr1 - 134984683 134985005 323 browser details YourSeq 207 81 846 3000 82.1% chr6 + 112867106 112867446 341 browser details YourSeq 205 568 846 3000 87.9% chr10 - 61797007 61797292 286 browser details YourSeq 203 563 842 3000 87.0% chr9 + 118657056 118657343 288 browser details YourSeq 202 538 848 3000 83.7% chrX + 12495161 12495469 309 browser details YourSeq 202 530 862 3000 85.9% chr14 + 118808124 118808471 348 browser details YourSeq 201 568 846 3000 86.9% chr5 - 30700923 30701210 288 Note: The 3000 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 3000 1 3000 3000 100.0% chr10 - 107081557 107084556 3000 browser details YourSeq 119 1540 1755 3000 86.5% chr12 + 85329336 85329551 216 browser details YourSeq 114 1536 1707 3000 89.6% chr2 - 58751224 58751397 174 browser details YourSeq 112 1538 1701 3000 86.0% chr6 + 53016967 53017152 186 browser details YourSeq 112 1536 1712 3000 90.0% chr4 + 131695665 131695842 178 browser details YourSeq 111 1536 1755 3000 82.7% chrX - 140092921 140093122 202 browser details YourSeq 106 1536 1709 3000 87.3% chr12 - 55713930 55714103 174 browser details YourSeq 105 1543 1755 3000 82.1% chr8 - 10983368 10983564 197 browser details YourSeq 104 1540 1707 3000 87.1% chr12 + 76783494 76783659 166 browser details YourSeq 103 1539 1755 3000 81.9% chr5 - 35674843 35675037 195 browser details YourSeq 102 1536 1712 3000 87.1% chr1 - 160388814 160388995 182 browser details YourSeq 100 1540 1679 3000 87.6% chr6 + 98079303 98079441 139 browser details YourSeq 97 1536 1761 3000 84.0% chr5 + 141813689 141813914 226 browser details YourSeq 97 1526 1692 3000 85.9% chr17 + 14856782 14856948 167 browser details YourSeq 96 1538 1755 3000 81.4% chr6 - 25781755 25781935 181 browser details YourSeq 95 1536 1696 3000 79.3% chrX + 76893361 76893515 155 browser details YourSeq 95 1540 1692 3000 88.0% chr2 + 148564294 148564447 154 browser details YourSeq 95 1538 1691 3000 87.5% chr10 + 61000518 61000672 155 browser details YourSeq 94 1536 1699 3000 91.7% chrX - 74354011 74354173 163 browser details YourSeq 94 1536 1694 3000 76.9% chr15 - 95146813 95146964 152 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Acss3 acyl-CoA synthetase short-chain family member 3 [ Mus musculus (house mouse) ] Gene ID: 380660, updated on 10-Oct-2019 Gene summary Official Symbol Acss3 provided by MGI Official Full Name acyl-CoA synthetase short-chain family member 3 provided by MGI Primary source MGI:MGI:2685720 See related Ensembl:ENSMUSG00000035948 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 Gm874; 8430416H19Rik Expression Biased expression in genital fat pad adult (RPKM 7.7), subcutaneous fat pad adult (RPKM 6.9) and 13 other tissues See Orthologs more human all Genomic context Location: 10; 10 D1 See Acss3 in Genome Data Viewer Exon count: 18 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (106933514..107123761, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (106373220..106560720, complement) Chromosome 10 - NC_000076.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Acss3 ENSMUSG00000035948 Description acyl-CoA synthetase short-chain family member 3 [Source:MGI Symbol;Acc:MGI:2685720] Gene Synonyms 8430416H19Rik, LOC380660 Location Chromosome 10: 106,933,517-107,123,668 reverse strand. GRCm38:CM001003.2 About this gene This gene has 3 transcripts (splice variants), 198 orthologues, 11 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Acss3-203 ENSMUST00000165067.8 4696 682aa ENSMUSP00000128209.1 Protein coding CCDS48688 Q14DH7 TSL:5 GENCODE basic APPRIS P1 Acss3-201 ENSMUST00000044668.4 2494 497aa ENSMUSP00000040823.4 Protein coding CCDS24159 Q14DH7 TSL:1 GENCODE basic Acss3-202 ENSMUST00000065567.5 1803 No protein - lncRNA - - TSL:1 210.15 kb Forward strand 106.95Mb 107.00Mb 107.05Mb 107.10Mb Genes Ppfia2-201 >protein coding (Comprehensive set... Ppfia2-203 >protein coding Ppfia2-204 >lncRNA Gm19007-201 >processed pseudogene Contigs AC134457.4 > AC121946.3 > Genes (Comprehensive set... < Acss3-203protein coding < Acss3-201protein coding < Gm18040-201processed pseudogene < Acss3-202lncRNA < Gm19181-201processed pseudogene Regulatory Build 106.95Mb 107.00Mb 107.05Mb 107.10Mb Reverse strand 210.15 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000165067 < Acss3-203protein coding Reverse strand 190.07 kb ENSMUSP00000128... Superfamily SSF56801 Pfam Acetyl-coenzyme A synthetase, N-terminal domain AMP-binding enzyme, C-terminal domain AMP-dependent synthetase/ligase PROSITE patterns AMP-binding, conserved site PANTHER PTHR43347:SF3 PTHR43347 Gene3D AMP-dependent synthetase-like superfamily 3.30.300.30 CDD cd05967 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 682 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.