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Mouse Abcd2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Abcd2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Abcd2 (NCBI Reference Sequence: NM_011994 ; Ensembl: ENSMUSG00000055782 ) is located on Mouse 15. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000069511). 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 Abcd2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-109D10 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: Mice homozygous for a disruption in this gene exhibit a late-onset cerebellar and sensory ataxia, loss of Purkinje cells, dorsal root ganglia cell degeneration, axonal degeneration in the spinal cord, and an accumulation of very long chain fatty acids.

Exon 2 starts from about 42.29% 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: 1654 bp, and the size of intron 2 for 3'-loxP site insertion: 6032 bp. The size of effective cKO region: ~681 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 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Abcd2 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(7181bp) | A(29.72% 2134) | C(19.65% 1411) | T(29.3% 2104) | G(21.33% 1532)

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% chr15 - 91189266 91192265 3000 browser details YourSeq 47 2857 2930 3000 90.8% chr13 + 11537266 11537338 73 browser details YourSeq 43 610 816 3000 95.9% chr2 - 11825109 11825422 314 browser details YourSeq 41 2880 2937 3000 81.3% chr2 - 135369113 135369166 54 browser details YourSeq 41 2877 2928 3000 90.4% chr11 + 46816216 46816269 54 browser details YourSeq 41 2877 2929 3000 91.5% chr1 + 119420115 119420169 55 browser details YourSeq 39 2883 2927 3000 95.4% chr5 + 116673201 116673245 45 browser details YourSeq 38 2875 2925 3000 93.1% chr17 + 15656169 15656672 504 browser details YourSeq 34 2887 2925 3000 97.3% chr2 - 106123807 106123847 41 browser details YourSeq 32 2889 2927 3000 94.8% chr13 + 39082219 39082283 65 browser details YourSeq 30 2888 2931 3000 85.8% chr13 + 6417741 6417782 42 browser details YourSeq 29 2881 2915 3000 94.0% chr13 + 7230465 7230499 35 browser details YourSeq 25 2877 2904 3000 84.7% chr2 + 157478972 157478997 26 browser details YourSeq 24 2898 2926 3000 92.9% chr17 + 4656806 4656836 31 browser details YourSeq 22 2752 2776 3000 95.9% chr13 + 43754070 43754096 27

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% chr15 - 91185585 91188584 3000 browser details YourSeq 704 1661 2994 3000 88.5% chr10 - 47985325 47987146 1822 browser details YourSeq 65 2322 2437 3000 90.2% chr11 - 102028799 102028919 121 browser details YourSeq 62 2311 2400 3000 89.8% chr14 - 54385480 54385569 90 browser details YourSeq 60 2295 2366 3000 92.8% chr15 - 70179566 70179637 72 browser details YourSeq 58 2311 2437 3000 91.5% chr12 - 45250673 45250804 132 browser details YourSeq 56 2312 2437 3000 93.9% chr14 + 6068749 6068877 129 browser details YourSeq 56 2311 2561 3000 89.1% chr13 + 95341852 95342109 258 browser details YourSeq 55 2311 2393 3000 88.8% chr13 - 27504334 27504422 89 browser details YourSeq 53 2758 2812 3000 98.2% chr1 + 106040852 106040906 55 browser details YourSeq 52 2319 2437 3000 92.0% chr14 - 10073895 10074018 124 browser details YourSeq 52 2311 2366 3000 96.5% chr1 - 136484867 136484922 56 browser details YourSeq 50 2311 2366 3000 94.7% chr12 - 69188219 69188274 56 browser details YourSeq 49 2322 2437 3000 94.6% chr14 - 5041234 5041349 116 browser details YourSeq 49 2322 2437 3000 94.6% chr14 + 6849668 6849783 116 browser details YourSeq 48 2311 2364 3000 94.5% chrX + 78063783 78063836 54 browser details YourSeq 47 2310 2366 3000 91.3% chr14 + 97825031 97825087 57 browser details YourSeq 46 2311 2366 3000 91.1% chr15 - 99412415 99412470 56 browser details YourSeq 45 2325 2387 3000 90.2% chr13 - 90842718 90842779 62 browser details YourSeq 44 2311 2366 3000 89.3% chr11 - 69971480 69971535 56

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 information: Abcd2 ATP-binding cassette, sub-family D (ALD), member 2 [ Mus musculus (house mouse) ] Gene ID: 26874, updated on 12-Aug-2019

Gene summary

Official Symbol Abcd2 provided by MGI Official Full Name ATP-binding cassette, sub-family D (ALD), member 2 provided by MGI Primary source MGI:MGI:1349467 See related Ensembl:ENSMUSG00000055782 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as ALDR; ABC39; ALDL1; ALDRP Summary The membrane-associated protein encoded by this gene is a member of the superfamily of ATP-binding cassette (ABC) Expression transporters. ABC transport various molecules across extra- and intra-cellular membranes. ABC are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the ALD subfamily, which is involved in peroxisomal import of fatty acids and/or fatty acyl-CoAs in the organelle. All known peroxisomal ABC transporters are half transporters which require a partner half transporter molecule to form a functional homodimeric or heterodimeric transporter. The function of this peroxisomal is unknown; however this protein is speculated to function as a dimerization partner of Abcd1 and/or other peroxisomal ABC transporters. Mutations in the human gene have been observed in patients with , a severe demyelinating disease. This gene has been identified as a candidate for a modifier gene, accounting for the extreme variation among adrenoleukodystrophy phenotypes. This gene is also a candidate for a complement group of , a genetically heterogeneous disorder of peroxisomal biogenesis. [provided by RefSeq, Jul 2008] Orthologs Biased expression in subcutaneous fat pad adult (RPKM 15.5), genital fat pad adult (RPKM 12.8) and 12 other tissues See more human all

Genomic context

Location: 15; 15 E3 See Abcd2 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (91145871..91192007, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (90976302..91022238, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 3 transcripts

Gene: Abcd2 ENSMUSG00000055782

Description ATP-binding cassette, sub-family D (ALD), member 2 [Source:MGI Symbol;Acc:MGI:1349467] Gene Synonyms ABC39, ALDL1, ALDR, adrenoleukodystrophy related Location Chromosome 15: 91,145,871-91,191,799 reverse strand. GRCm38:CM001008.2 About this gene This gene has 3 transcripts (splice variants), 205 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 23 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Abcd2-201 ENSMUST00000069511.7 5532 741aa ENSMUSP00000068940.6 Protein coding CCDS27760 A0A0R4J0U5 TSL:1 GENCODE basic APPRIS P1

Abcd2-203 ENSMUST00000230461.1 528 No protein - Retained intron - - -

Abcd2-202 ENSMUST00000229909.1 320 No protein - lncRNA - - -

65.93 kb Forward strand

91.14Mb 91.16Mb 91.18Mb 91.20Mb Genes CN725425-202 >protein coding (Comprehensive set...

Contigs < AC113102.8 Genes (Comprehensive set... < Abcd2-201protein coding

< Abcd2-202lncRNA < Abcd2-203retained intron

Regulatory Build

91.14Mb 91.16Mb 91.18Mb 91.20Mb Reverse strand 65.93 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000069511

< Abcd2-201protein coding

Reverse strand 45.93 kb

ENSMUSP00000068... Low complexity (Seg) Superfamily ABC transporter type 1, transmembrane domain superfamily P-loop containing nucleoside triphosphate hydrolase

SMART AAA+ ATPase domain

Pfam ABC transporter type 1, transmembrane domain ABC transporter-like

PROSITE profiles ABC transporter type 1, transmembrane domain ABC transporter-like

PROSITE patterns ABC transporter, conserved site PANTHER ATP-binding cassette sub-family D member 2

PTHR11384 Gene3D 3.40.50.300

CDD cd03223

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 741

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

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