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Mouse Acsm3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Acsm3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Acsm3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Acsm3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Acsm3 gene (NCBI Reference Sequence: NM_016870 ; Ensembl: ENSMUSG00000030935 ) is located on Mouse chromosome 7. 16 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000106528). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Acsm3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-202C8 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: Homozygous null mice are viable and fertile with normal kidney function and morphology and blood pressure similar to wild-type on either a regular or high salt diet. Exon 6 starts from about 23.74% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 4713 bp, and the size of intron 6 for 3'-loxP site insertion: 617 bp. The size of effective cKO region: ~708 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 6 7 8 9 16 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Acsm3 Homology arm cKO region loxP site Page 2 of 8 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(7208bp) | A(30.79% 2219) | C(18.84% 1358) | T(28.34% 2043) | G(22.03% 1588) 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 8 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% chr7 + 119770439 119773438 3000 browser details YourSeq 131 1526 1976 3000 84.7% chr11 - 50549341 50549809 469 browser details YourSeq 121 1654 1980 3000 82.4% chr11 - 11350711 11351049 339 browser details YourSeq 119 1646 1972 3000 86.5% chr8 + 89132032 89132417 386 browser details YourSeq 115 1650 2007 3000 84.0% chr1 + 79187010 79187373 364 browser details YourSeq 112 1652 2013 3000 85.0% chr7 - 49011430 49011786 357 browser details YourSeq 109 1659 1943 3000 86.1% chr6 - 127968865 127969154 290 browser details YourSeq 107 1655 1950 3000 79.9% chr1 + 156984660 156984959 300 browser details YourSeq 100 1650 2009 3000 86.3% chr1 - 119903718 119904155 438 browser details YourSeq 98 1655 1930 3000 84.1% chr4 + 72107245 72107542 298 browser details YourSeq 91 1524 1850 3000 84.9% chr5 - 75686268 75686852 585 browser details YourSeq 89 1675 1945 3000 77.4% chr12 + 11501370 11501641 272 browser details YourSeq 88 1629 1951 3000 78.8% chr18 - 78040017 78040331 315 browser details YourSeq 88 1657 1809 3000 86.2% chr19 + 4844105 4844257 153 browser details YourSeq 87 1655 1862 3000 89.9% chr17 - 47129417 47129624 208 browser details YourSeq 87 1587 1878 3000 76.2% chr13 + 31662341 31662631 291 browser details YourSeq 78 1655 1850 3000 85.0% chr7 - 64542720 64542912 193 browser details YourSeq 76 1650 1865 3000 88.8% chr13 - 65853090 65853306 217 browser details YourSeq 76 1650 1865 3000 88.8% chr13 + 66073201 66073417 217 browser details YourSeq 73 1694 1972 3000 87.7% chrX - 96203291 96203587 297 Note: The 3000 bp section upstream of Exon 6 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% chr7 + 119774147 119777146 3000 browser details YourSeq 185 581 813 3000 90.5% chr14 - 26366751 26366985 235 browser details YourSeq 182 581 822 3000 90.8% chr1 - 95161858 95162117 260 browser details YourSeq 179 583 823 3000 91.0% chr9 - 73135480 73136059 580 browser details YourSeq 178 581 820 3000 90.5% chr9 - 118263044 118263292 249 browser details YourSeq 178 582 822 3000 87.9% chr16 + 72722807 72723052 246 browser details YourSeq 177 581 816 3000 88.8% chr10 - 126364128 126364365 238 browser details YourSeq 177 581 819 3000 90.6% chr5 + 107326450 107326685 236 browser details YourSeq 177 581 823 3000 90.9% chr5 + 99408263 99408505 243 browser details YourSeq 176 581 822 3000 86.8% chr14 - 20242163 20242397 235 browser details YourSeq 176 581 872 3000 86.8% chr5 + 90781497 90782170 674 browser details YourSeq 175 595 875 3000 89.1% chr16 + 56395130 56395549 420 browser details YourSeq 175 581 823 3000 91.2% chr14 + 70868995 70869241 247 browser details YourSeq 174 581 817 3000 90.4% chrX - 57160201 57160438 238 browser details YourSeq 174 581 810 3000 87.7% chr15 - 31136116 31136330 215 browser details YourSeq 174 581 821 3000 86.7% chr14 - 60800401 60800634 234 browser details YourSeq 174 581 821 3000 86.9% chr19 + 59303595 59303827 233 browser details YourSeq 173 581 810 3000 91.1% chr2 - 29608880 29609112 233 browser details YourSeq 173 581 830 3000 86.1% chr1 - 186853320 186853566 247 browser details YourSeq 172 581 804 3000 91.1% chr18 + 64871474 64871973 500 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Acsm3 acyl-CoA synthetase medium-chain family member 3 [ Mus musculus (house mouse) ] Gene ID: 20216, updated on 10-Oct-2019 Gene summary Official Symbol Acsm3 provided by MGI Official Full Name acyl-CoA synthetase medium-chain family member 3 provided by MGI Primary source MGI:MGI:99538 See related Ensembl:ENSMUSG00000030935 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 Sa; Sah Expression Broad expression in liver E18 (RPKM 13.3), liver adult (RPKM 7.2) and 18 other tissues See more Orthologs human all Genomic context Location: 7; 7 F2 See Acsm3 in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (119760883..119784896) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (126904437..126928410) Chromosome 7 - NC_000073.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Acsm3 ENSMUSG00000030935 Description acyl-CoA synthetase medium-chain family member 3 [Source:MGI Symbol;Acc:MGI:99538] Gene Synonyms Sa, Sah Location Chromosome 7: 119,760,923-119,787,513 forward strand. GRCm38:CM001000.2 About this gene This gene has 8 transcripts (splice variants), 159 orthologues, 11 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 Acsm3-204 ENSMUST00000106528.7 2755 580aa ENSMUSP00000102138.1 Protein coding CCDS21786 Q3UNX5 TSL:1 GENCODE basic APPRIS P1 Acsm3-202 ENSMUST00000106526.1 2704 580aa ENSMUSP00000102136.1 Protein coding CCDS21786 Q3UNX5 TSL:5 GENCODE basic APPRIS P1 Acsm3-201 ENSMUST00000063770.9 2651 580aa ENSMUSP00000068803.3 Protein coding CCDS21786 Q3UNX5 TSL:1 GENCODE basic APPRIS P1 Acsm3-203 ENSMUST00000106527.7 2557 580aa ENSMUSP00000102137.1 Protein coding CCDS21786 Q3UNX5 TSL:1 GENCODE basic APPRIS P1 Acsm3-205 ENSMUST00000106529.7 3295 622aa ENSMUSP00000102139.1 Protein coding - Q3UNX5 TSL:1 GENCODE basic Acsm3-207 ENSMUST00000149766.1 1325 No protein - Retained intron - - TSL:1 Acsm3-206 ENSMUST00000149598.1 488 No protein - Retained intron - - TSL:2 Acsm3-208 ENSMUST00000154828.1 229 No protein - Retained intron - - TSL:5 Page 6 of 8 https://www.alphaknockout.com 46.59 kb Forward strand 119.76Mb 119.77Mb 119.78Mb 119.79Mb Genes (Comprehensive set... Gm44966-201 >processed pseudogene Acsm3-202 >protein coding Rexo5-204 >nonsense mediated decay Acsm3-204 >protein coding Rexo5-205 >retained intron Acsm3-203 >protein coding Rexo5-201 >protein coding Acsm3-205 >protein coding Rexo5-203 >protein coding Acsm3-201 >protein coding Rexo5-202 >protein coding Acsm3-207 >retained intron Acsm3-206 >retained intron Acsm3-208 >retained intron Contigs < AC164158.3 Genes < Eri2-203protein coding (Comprehensive set... < Eri2-202protein coding < Eri2-204retained intron < Eri2-201nonsense mediated decay < Eri2-207protein coding < Eri2-206nonsense mediated decay < Eri2-205retained intron Regulatory Build 119.76Mb 119.77Mb 119.78Mb 119.79Mb Reverse strand 46.59 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000106528 23.97 kb Forward strand Acsm3-204 >protein coding ENSMUSP00000102... Superfamily SSF56801 Pfam AMP-dependent synthetase/ligase AMP-binding enzyme, C-terminal domain PROSITE patterns AMP-binding, conserved site PANTHER PTHR43605:SF7 PTHR43605 Gene3D AMP-dependent synthetase-like superfamily 3.30.300.310 CDD cd05928 All sequence SNPs/i..
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