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Mouse Acaa2 Knockout Project (CRISPR/Cas9)

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Mouse Acaa2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Acaa2 Knockout Project (CRISPR/Cas9) Objective: To create a Acaa2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Acaa2 gene (NCBI Reference Sequence: NM_177470 ; Ensembl: ENSMUSG00000036880 ) is located on Mouse chromosome 18. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000041053). 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 1.43% of the coding region. Exon 2~4 covers 34.68% of the coding region. The size of effective KO region: ~6285 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 10 Legends Exon of mouse Acaa2 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 1727 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(29.0% 580) | C(19.9% 398) | T(31.9% 638) | G(19.2% 384) 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(1727bp) | A(30.34% 524) | C(20.21% 349) | T(29.65% 512) | G(19.8% 342) Note: The 1727 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% chr18 + 74785120 74787119 2000 browser details YourSeq 61 665 767 2000 86.8% chr18 - 74786304 74786412 109 browser details YourSeq 53 891 1181 2000 81.4% chr10 - 128355921 128356203 283 browser details YourSeq 45 1120 1182 2000 82.3% chr18 - 3696355 3696416 62 browser details YourSeq 45 1123 1190 2000 84.4% chr11 - 71844922 71844989 68 browser details YourSeq 44 1129 1192 2000 79.7% chr5 + 140281542 140281598 57 browser details YourSeq 43 1120 1174 2000 85.2% chrX + 160248770 160248823 54 browser details YourSeq 41 1129 1190 2000 84.5% chr10 - 88690176 88690237 62 browser details YourSeq 41 359 399 2000 100.0% chr4 + 101675116 101675156 41 browser details YourSeq 37 1118 1178 2000 82.0% chr1 + 56545031 56545090 60 browser details YourSeq 36 1136 1192 2000 74.0% chr10 - 78225701 78225751 51 browser details YourSeq 36 1123 1178 2000 82.2% chr18 + 24782084 24782139 56 browser details YourSeq 35 1129 1181 2000 75.0% chr11 - 72680326 72680369 44 browser details YourSeq 35 1129 1181 2000 78.9% chr10 - 102793371 102793422 52 browser details YourSeq 35 1134 1190 2000 76.8% chr10 - 34186398 34186453 56 browser details YourSeq 35 1134 1176 2000 90.7% chr1 + 118683525 118683567 43 browser details YourSeq 34 1127 1176 2000 84.0% chr4 - 11259264 11259313 50 browser details YourSeq 34 1134 1175 2000 90.5% chr11 + 61856945 61856986 42 browser details YourSeq 34 1124 1192 2000 66.7% chr10 + 120140012 120140074 63 browser details YourSeq 34 1130 1181 2000 78.5% chr10 + 112583900 112583950 51 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 1727 1 1727 1727 100.0% chr18 + 74793405 74795131 1727 browser details YourSeq 461 1048 1614 1727 90.8% chr9 - 80059727 80060290 564 browser details YourSeq 442 1043 1615 1727 88.3% chr3 + 25167813 25168373 561 browser details YourSeq 439 1070 1617 1727 90.5% chrX - 18390724 18783468 392745 browser details YourSeq 437 1051 1617 1727 90.5% chrX + 65458833 65459396 564 browser details YourSeq 437 1051 1617 1727 89.5% chr8 + 3550322 3550883 562 browser details YourSeq 436 1053 1617 1727 88.5% chr18 + 4951111 4951672 562 browser details YourSeq 433 797 1610 1727 87.2% chr15 - 66419254 66419829 576 browser details YourSeq 432 1051 1617 1727 88.3% chr8 - 65210496 65211052 557 browser details YourSeq 429 1051 1617 1727 88.5% chr4 - 61839571 61964596 125026 browser details YourSeq 426 1051 1617 1727 88.9% chr4 - 60226711 60506675 279965 browser details YourSeq 422 1051 1617 1727 88.1% chr4 - 61599821 62094102 494282 browser details YourSeq 420 1051 1617 1727 88.3% chr4 - 61678335 61786770 108436 browser details YourSeq 419 1074 1617 1727 88.4% chr11 - 103481238 103481778 541 browser details YourSeq 418 1051 1617 1727 86.4% chr12 + 104288905 104289462 558 browser details YourSeq 415 1051 1612 1727 89.7% chr10 + 46818135 46818701 567 browser details YourSeq 414 1051 1617 1727 88.6% chr4 - 61443686 61925582 481897 browser details YourSeq 414 1083 1617 1727 90.0% chr10 - 26633889 26634435 547 browser details YourSeq 413 1051 1617 1727 87.8% chr4 - 61232230 61444247 212018 browser details YourSeq 411 1051 1617 1727 86.8% chr4 - 62057744 62058307 564 Note: The 1727 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: Acaa2 acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase) [ Mus musculus (house mouse) ] Gene ID: 52538, updated on 10-Oct-2019 Gene summary Official Symbol Acaa2 provided by MGI Official Full Name acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase) provided by MGI Primary source MGI:MGI:1098623 See related Ensembl:ENSMUSG00000036880 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 AI255831; AI265397; D18Ertd240e; 0610011L04Rik Expression Broad expression in liver E18 (RPKM 299.0), heart adult (RPKM 261.9) and 19 other tissues See more Orthologs human all Genomic context Location: 18 E2; 18 50.76 cM See Acaa2 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (74779212..74806207) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (74938866..74965861) Chromosome 18 - NC_000084.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Acaa2 ENSMUSG00000036880 Description acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase) [Source:MGI Symbol;Acc:MGI:1098623] Gene Synonyms 0610011L04Rik, D18Ertd240e Location Chromosome 18: 74,779,197-74,806,208 forward strand. GRCm38:CM001011.2 About this gene This gene has 2 transcripts (splice variants), 209 orthologues, 6 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Acaa2-201 ENSMUST00000041053.10 1516 397aa ENSMUSP00000037348.9 Protein coding CCDS29342 Q8BWT1 TSL:1 GENCODE basic APPRIS P1 Acaa2-202 ENSMUST00000237306.1 504 103aa ENSMUSP00000157638.1 Protein coding - A0A494B9J2 CDS 5' incomplete 47.01 kb Forward strand 74.77Mb 74.78Mb 74.79Mb 74.80Mb 74.81Mb Genes (Comprehensive set... Myo5b-201 >protein coding Acaa2-201 >protein coding Myo5b-203 >protein coding Acaa2-202 >protein coding Myo5b-210 >retained intron Contigs AC148002.3 > Genes < Scarna17-201scaRNA (Comprehensive set... < Gm23119-201scaRNA Regulatory Build 74.77Mb 74.78Mb 74.79Mb 74.80Mb 74.81Mb Reverse strand 47.01 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 processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000041053 27.01 kb Forward strand Acaa2-201 >protein coding ENSMUSP00000037... Low complexity (Seg) TIGRFAM Thiolase Superfamily Thiolase-like Pfam Thiolase, N-terminal Thiolase, C-terminal PROSITE patterns Thiolase, acyl-enzyme intermediate active site Thiolase, conserved site Thiolase, active site PIRSF Thiolase PANTHER PTHR18919 PTHR18919:SF145 Gene3D Thiolase-like CDD Thiolase All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 397 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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