https://www.alphaknockout.com

Mouse Acot6 Knockout Project (CRISPR/Cas9)

Objective: To create a Acot6 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Acot6 (NCBI Reference Sequence: NM_172580 ; Ensembl: ENSMUSG00000043487 ) is located on Mouse 12. 3 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 3 (Transcript: ENSMUST00000056822). Exon 1~3 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 knock-out allele exhibit no detectable phenotypic abnormalities.

Exon 1 starts from about 0.08% of the coding region. Exon 1~3 covers 100.0% of the coding region. The size of effective KO region: ~8565 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3

Legends Exon of mouse Acot6 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of start codon 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of stop codon 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(26.1% 522) | C(21.75% 435) | T(29.3% 586) | G(22.85% 457)

Note: The 2000 bp section upstream of start codon 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(25.6% 512) | C(24.0% 480) | T(31.0% 620) | G(19.4% 388)

Note: The 2000 bp section downstream of stop codon 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr12 + 84098973 84100972 2000 browser details YourSeq 507 593 1434 2000 92.6% chr12 + 84635223 84636237 1015 browser details YourSeq 473 569 1395 2000 91.9% chr19 + 56525779 56526650 872 browser details YourSeq 440 569 1395 2000 90.6% chr1_GL456213_random + 34045 34917 873 browser details YourSeq 425 554 1432 2000 87.8% chr12 - 86238369 86239012 644 browser details YourSeq 410 549 1401 2000 87.4% chr11 - 78719934 78720785 852 browser details YourSeq 397 686 1387 2000 88.8% chr9 - 57319246 57319964 719 browser details YourSeq 388 551 1392 2000 86.0% chr17 - 55872548 55873203 656 browser details YourSeq 385 551 1412 2000 86.5% chr9 - 96067688 96068651 964 browser details YourSeq 385 551 1395 2000 83.6% chr1 - 94074669 94075325 657 browser details YourSeq 379 545 1237 2000 88.0% chr19 + 38370574 38371657 1084 browser details YourSeq 376 624 1416 2000 86.0% chr3 + 32807053 32807630 578 browser details YourSeq 359 551 1409 2000 85.2% chr1 - 72638686 72639309 624 browser details YourSeq 357 551 1420 2000 83.6% chr6 - 35157114 35157766 653 browser details YourSeq 355 555 1436 2000 87.1% chr8 + 25906670 25907406 737 browser details YourSeq 354 551 1435 2000 83.5% chr7 + 92703344 92704033 690 browser details YourSeq 353 555 1395 2000 86.6% chr1 + 174493303 174493955 653 browser details YourSeq 352 707 1420 2000 84.9% chr6 + 40026869 40027404 536 browser details YourSeq 352 557 1401 2000 84.6% chr1 + 98838608 98839235 628 browser details YourSeq 347 686 1410 2000 85.0% chr13 + 24735610 24736132 523

Note: The 2000 bp section upstream of start codon 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% chr12 + 84109538 84111537 2000 browser details YourSeq 222 289 682 2000 84.5% chr15 + 58883139 58883538 400 browser details YourSeq 200 297 711 2000 82.1% chr6 - 66510950 66511358 409 browser details YourSeq 199 266 723 2000 88.2% chr6 - 5666020 5666490 471 browser details YourSeq 185 286 620 2000 84.4% chr18 + 42504265 42504591 327 browser details YourSeq 183 286 620 2000 82.8% chr4 + 41439400 41439730 331 browser details YourSeq 178 286 680 2000 86.9% chr10 - 45255887 45256288 402 browser details YourSeq 167 300 620 2000 88.5% chrX - 157630290 157630614 325 browser details YourSeq 163 308 620 2000 82.9% chr10 - 41332710 41333016 307 browser details YourSeq 151 266 620 2000 81.4% chrX + 60460969 60461337 369 browser details YourSeq 151 286 680 2000 83.0% chr8 + 33719267 33719662 396 browser details YourSeq 127 286 706 2000 81.9% chr13 - 8893345 8893739 395 browser details YourSeq 119 294 617 2000 77.2% chr15 + 19321211 19321508 298 browser details YourSeq 118 308 488 2000 88.9% chr1 + 156344564 156344751 188 browser details YourSeq 117 1838 2000 2000 91.0% chr9 - 28819130 28819323 194 browser details YourSeq 116 286 591 2000 91.0% chr3 + 131313641 131314322 682 browser details YourSeq 114 1838 2000 2000 89.3% chr16 - 92245820 92245982 163 browser details YourSeq 113 1837 1985 2000 90.0% chr3 - 52253013 52253195 183 browser details YourSeq 112 1838 2000 2000 90.1% chr5 - 30736148 30736330 183 browser details YourSeq 112 351 581 2000 81.5% chr13 - 67288228 67288463 236

Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Acot6 acyl-CoA 6 [ Mus musculus (house mouse) ] Gene ID: 217700, updated on 12-Aug-2019

Gene summary

Official Symbol Acot6 provided by MGI Official Full Name acyl-CoA thioesterase 6 provided by MGI Primary source MGI:MGI:1921287 See related Ensembl:ENSMUSG00000043487 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 BE688602; A330054B07; 4632408A20Rik Expression Ubiquitous expression in ovary adult (RPKM 11.9), kidney adult (RPKM 8.5) and 27 other tissues See more Orthologs human all

Genomic context

Location: 12; 12 D1 See Acot6 in Genome Data Viewer Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (84100654..84109783)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (85441604..85450733)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 2 transcripts

Gene: Acot6 ENSMUSG00000043487

Description acyl-CoA thioesterase 6 [Source:MGI Symbol;Acc:MGI:1921287] Gene Synonyms 4632408A20Rik Location Chromosome 12: 84,100,654-84,111,349 forward strand. GRCm38:CM001005.2 About this gene This gene has 2 transcripts (splice variants), 361 orthologues, 9 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

Acot6-201 ENSMUST00000056822.3 3388 419aa ENSMUSP00000056131.3 Protein coding CCDS26038 B2RTE4 Q32Q92 TSL:1 GENCODE basic APPRIS P1

Acot6-202 ENSMUST00000222921.1 3622 176aa ENSMUSP00000152129.1 Protein coding - Q32Q92 TSL:NA GENCODE basic

30.70 kb Forward strand

84.10Mb 84.11Mb 84.12Mb Acot6-201 >protein coding Dnal1-202 >protein coding (Comprehensive set...

Acot6-202 >protein coding Dnal1-206 >lncRNA

Dnal1-201 >protein coding

Dnal1-207 >nonsense mediated decay

Dnal1-203 >protein coding

Contigs < AC125071.3 Regulatory Build

84.10Mb 84.11Mb 84.12Mb Reverse strand 30.70 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000056822

10.70 kb Forward strand

Acot6-201 >protein coding

ENSMUSP00000056... Low complexity (Seg) Superfamily Alpha/Beta hydrolase fold

Pfam BAAT/Acyl-CoA thioester hydrolase C-terminal

Acyl-CoA thioester hydrolase/bile acid-CoA N-acetyltransferase PIRSF Acyl-CoA thioesterase, long chain PANTHER PTHR10824

PTHR10824:SF17 Gene3D Acyl-CoA thioester hydrolase/BAAT, N-terminal

Alpha/Beta hydrolase fold

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 419

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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