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Mouse Acbd3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Acbd3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Acbd3 gene (NCBI Reference Sequence: NM_133225 ; Ensembl: ENSMUSG00000026499 ) is located on Mouse chromosome 1. 8 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000027780). 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 Acbd3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-141O11 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 17.65% 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: 6787 bp, and the size of intron 2 for 3'-loxP site insertion: 1069 bp. The size of effective cKO region: ~642 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 3 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Acbd3 Homology arm cKO region loxP site

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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(7142bp) | A(26.69% 1906) | C(19.53% 1395) | T(32.44% 2317) | G(21.34% 1524)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr1 + 180729917 180732916 3000 browser details YourSeq 270 1562 2076 3000 93.9% chr17 - 17358971 17799790 440820 browser details YourSeq 253 1759 2070 3000 92.7% chr8 - 107434743 107621852 187110 browser details YourSeq 249 1750 2079 3000 93.8% chr2 - 34780154 34780567 414 browser details YourSeq 233 1757 2078 3000 91.9% chr10 - 100554748 100555281 534 browser details YourSeq 231 1761 2079 3000 91.1% chr5 - 115499898 115500256 359 browser details YourSeq 209 1765 2078 3000 90.1% chr4 + 86768129 86768732 604 browser details YourSeq 206 1759 2139 3000 91.6% chr15 + 100287156 100287577 422 browser details YourSeq 181 759 1893 3000 86.6% chr4 - 118819904 118986797 166894 browser details YourSeq 166 1757 2082 3000 92.0% chr5 + 33315571 33316051 481 browser details YourSeq 154 1594 1932 3000 86.5% chr11 + 8760194 8760429 236 browser details YourSeq 148 1910 2091 3000 96.3% chr6 + 28786146 28786373 228 browser details YourSeq 143 1743 1911 3000 92.9% chr2 - 3299509 3299679 171 browser details YourSeq 143 1757 1924 3000 93.4% chr10 - 85639175 85639345 171 browser details YourSeq 141 1910 2079 3000 94.5% chr13 + 62183578 62183876 299 browser details YourSeq 140 1920 2080 3000 94.4% chr12 + 73628406 73628729 324 browser details YourSeq 137 1765 1923 3000 93.8% chr8 + 33630676 33630844 169 browser details YourSeq 137 1752 1907 3000 94.8% chr16 + 21639545 21639709 165 browser details YourSeq 137 1761 1976 3000 91.0% chr10 + 122996696 122997726 1031 browser details YourSeq 135 1930 2078 3000 96.0% chrX - 75064037 75064555 519

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% chr1 + 180733559 180736558 3000 browser details YourSeq 225 1 342 3000 90.4% chr18 - 84905339 84905907 569 browser details YourSeq 222 1 265 3000 92.4% chr17 - 32328051 32328315 265 browser details YourSeq 222 1 262 3000 92.4% chr13 - 103792624 103792885 262 browser details YourSeq 221 1 263 3000 92.1% chr4 - 116867381 116867643 263 browser details YourSeq 221 5 276 3000 92.1% chr17 + 43014314 43014858 545 browser details YourSeq 220 1 265 3000 92.7% chr3 + 9039232 9039513 282 browser details YourSeq 219 9 442 3000 92.0% chrX + 56393345 56393794 450 browser details YourSeq 219 1 267 3000 91.7% chr19 + 5962056 5962339 284 browser details YourSeq 219 1 266 3000 92.0% chr10 + 85542965 85543266 302 browser details YourSeq 218 1 269 3000 91.4% chr12 - 69205079 69205362 284 browser details YourSeq 218 1 482 3000 93.0% chr11 + 79137631 79138127 497 browser details YourSeq 217 1 268 3000 91.3% chr2 - 144469044 144469331 288 browser details YourSeq 217 2 264 3000 92.6% chr12 - 72977563 72977862 300 browser details YourSeq 217 1 265 3000 90.5% chr4 + 148116225 148116488 264 browser details YourSeq 216 1 266 3000 91.2% chr8 - 121508450 121508767 318 browser details YourSeq 214 2 268 3000 91.5% chr5 - 107607559 107607855 297 browser details YourSeq 213 1 267 3000 91.2% chr4 - 128757096 128757380 285 browser details YourSeq 213 1 264 3000 91.1% chr10 - 63356490 63356785 296 browser details YourSeq 213 2 264 3000 90.9% chr8 + 104016098 104016360 263

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Acbd3 acyl-Coenzyme A binding domain containing 3 [ Mus musculus (house mouse) ] Gene ID: 170760, updated on 12-Aug-2019

Gene summary

Official Symbol Acbd3 provided by MGI Official Full Name acyl-Coenzyme A binding domain containing 3 provided by MGI Primary source MGI:MGI:2181074 See related Ensembl:ENSMUSG00000026499 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 Pap7; 60kDa; GCP60; GOLPH1; Gocap1; D1Ertd10e; 8430407O11Rik Expression Ubiquitous expression in placenta adult (RPKM 17.7), limb E14.5 (RPKM 13.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: 1 H4; 1 84.46 cM See Acbd3 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (180726043..180754204)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (182656174..182684335)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 2 transcripts

Gene: Acbd3 ENSMUSG00000026499

Description acyl-Coenzyme A binding domain containing 3 [Source:MGI Symbol;Acc:MGI:2181074] Gene Synonyms 8430407O11Rik, D1Ertd10e, Gocap1, Pap7 Location Chromosome 1: 180,726,043-180,754,204 forward strand. GRCm38:CM000994.2 About this gene This gene has 2 transcripts (splice variants), 213 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Acbd3-201 ENSMUST00000027780.5 3471 525aa ENSMUSP00000027780.4 Protein coding CCDS15572 A0A0R4J079 TSL:1 GENCODE basic APPRIS P1

Acbd3-202 ENSMUST00000192420.1 3569 No protein - Retained intron - - TSL:1

48.16 kb Forward strand

180.72Mb 180.73Mb 180.74Mb 180.75Mb 180.76Mb Genes Gm8101-201 >processed pseudogene (Comprehensive set...

Acbd3-201 >protein coding

Acbd3-202 >retained intron

Contigs AC167020.3 >

Genes < Gm37768-201lncRNA < Gm38293-201lncRNA (Comprehensive set...

Regulatory Build

180.72Mb 180.73Mb 180.74Mb 180.75Mb 180.76Mb Reverse strand 48.16 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene processed transcript RNA gene

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Transcript: ENSMUST00000027780

28.16 kb Forward strand

Acbd3-201 >protein coding

ENSMUSP00000027... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Acyl-CoA binding protein superfamily GOLD domain superfamily

Pfam Acyl-CoA-binding protein, ACBP GOLD domain

PROSITE profiles Acyl-CoA-binding protein, ACBP GOLD domain

PROSITE patterns Acyl-CoA-binding protein, ACBP, conserved site PANTHER PTHR22973:SF11

PTHR22973 Gene3D FERM/acyl-CoA-binding protein superfamily 2.60.120.680

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 525

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

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