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Mouse Abca5 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Abca5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Abca5 gene (NCBI Reference Sequence: NM_147219 ; Ensembl: ENSMUSG00000018800 ) is located on Mouse chromosome 11. 39 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 39 (Transcript: ENSMUST00000043961). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Abca5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-198N23 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 knock-out allele exhibit exophthalmos, tremors and collapse of the thyroid gland, and develop a dilated cardiomyopathy with large thrombi due to depression of the cardiac function. Severe edema, liver injury and premature death appear to be sensitive to genetic background.

Exon 2 starts from about 0.04% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 8228 bp, and the size of intron 3 for 3'-loxP site insertion: 1010 bp. The size of effective cKO region: ~2007 bp. The cKO 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 4 39 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Abca5 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(8507bp) | A(27.02% 2299) | C(18.94% 1611) | T(33.7% 2867) | G(20.34% 1730)

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% chr11 - 110329489 110332488 3000 browser details YourSeq 142 2755 2911 3000 95.6% chr5 - 130085150 130085307 158 browser details YourSeq 140 2755 2910 3000 94.1% chr7 - 65872873 65873026 154 browser details YourSeq 139 2750 2911 3000 93.2% chr4 - 109062952 109063113 162 browser details YourSeq 139 2755 2906 3000 96.1% chr1 + 6189653 6189806 154 browser details YourSeq 138 2755 2925 3000 91.3% chr10 + 5624043 5624211 169 browser details YourSeq 137 2701 2903 3000 96.7% chr10 - 107055727 107056050 324 browser details YourSeq 137 2755 2911 3000 96.7% chr7 + 19680215 19680372 158 browser details YourSeq 137 2758 2928 3000 93.1% chr2 + 68973390 68973565 176 browser details YourSeq 137 2755 2910 3000 94.3% chr12 + 86798606 86798763 158 browser details YourSeq 137 2756 2911 3000 95.5% chr1 + 34796871 34797027 157 browser details YourSeq 136 2755 2907 3000 94.8% chr8 - 22873058 22873212 155 browser details YourSeq 136 2757 2910 3000 94.8% chr6 - 113308949 113309102 154 browser details YourSeq 136 2755 2910 3000 92.8% chr12 + 3752958 3753111 154 browser details YourSeq 135 2755 2911 3000 94.2% chr3 - 86619070 86619226 157 browser details YourSeq 135 2755 2910 3000 94.2% chr2 - 181858810 181858966 157 browser details YourSeq 135 2758 2910 3000 94.8% chr14 + 64968420 64968586 167 browser details YourSeq 134 2755 2911 3000 93.0% chr9 - 113804864 113805022 159 browser details YourSeq 134 2747 2906 3000 92.5% chr2 - 84732926 84733085 160 browser details YourSeq 134 2755 2910 3000 92.1% chr17 - 71368834 71368987 154

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% chr11 - 110324482 110327481 3000 browser details YourSeq 216 2599 2988 3000 89.3% chr2 + 153894020 153894418 399 browser details YourSeq 194 2599 2972 3000 87.3% chr12 + 30764350 30764724 375 browser details YourSeq 190 2685 2993 3000 89.6% chr3 - 129344024 129344335 312 browser details YourSeq 190 2599 2985 3000 91.4% chr2 - 159138552 159138973 422 browser details YourSeq 189 2599 2988 3000 86.0% chr6 - 30892367 30892769 403 browser details YourSeq 185 2637 2988 3000 89.6% chr9 - 107019205 107019555 351 browser details YourSeq 185 2599 2988 3000 91.2% chr2 + 101398708 101399100 393 browser details YourSeq 181 2648 2980 3000 82.7% chr3 - 130613313 130613626 314 browser details YourSeq 181 2721 2985 3000 87.5% chr11 + 77232304 77232561 258 browser details YourSeq 178 2599 2982 3000 88.4% chr11 - 11531984 11532371 388 browser details YourSeq 177 2648 2988 3000 87.7% chr13 - 51235890 51236233 344 browser details YourSeq 177 2648 2988 3000 90.1% chr9 + 115702569 115702914 346 browser details YourSeq 176 2666 2988 3000 86.8% chr4 + 87778370 87778687 318 browser details YourSeq 175 2599 2975 3000 88.9% chr3 - 15121047 15121425 379 browser details YourSeq 175 2704 2996 3000 86.9% chrX + 85159113 85159405 293 browser details YourSeq 174 2648 3000 3000 89.6% chrX - 94442381 94442741 361 browser details YourSeq 174 2648 2988 3000 88.5% chrX + 48335268 48335615 348 browser details YourSeq 172 2599 2967 3000 91.8% chr10 - 22503445 22503835 391 browser details YourSeq 171 2698 2980 3000 87.8% chr14 + 104159010 104159288 279

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Abca5 ATP-binding cassette, sub-family A (ABC1), member 5 [ Mus musculus (house mouse) ] Gene ID: 217265, updated on 12-Aug-2019

Gene summary

Official Symbol Abca5 provided by MGI Official Full Name ATP-binding cassette, sub-family A (ABC1), member 5 provided by MGI Primary source MGI:MGI:2386607 See related Ensembl:ENSMUSG00000018800 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 ABC13; mKIAA1888; B930033A02Rik Expression Broad expression in cerebellum adult (RPKM 4.0), cortex adult (RPKM 3.1) and 19 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 E1 See Abca5 in Genome Data Viewer

Exon count: 39

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (110269369..110337723, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (110130683..110199030, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 5 transcripts

Gene: Abca5 ENSMUSG00000018800

Description ATP-binding cassette, sub-family A (ABC1), member 5 [Source:MGI Symbol;Acc:MGI:2386607] Gene Synonyms ABC13, B930033A02Rik Location Chromosome 11: 110,269,369-110,337,716 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 215 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Abca5-201 ENSMUST00000043961.11 8231 1642aa ENSMUSP00000047927.5 Protein coding CCDS25591 Q8K448 TSL:1 GENCODE basic APPRIS P1

Abca5-202 ENSMUST00000124714.7 3829 1194aa ENSMUSP00000120708.1 Protein coding - A2AEP5 CDS 3' incomplete TSL:1

Abca5-204 ENSMUST00000134721.1 610 181aa ENSMUSP00000118328.1 Protein coding - A2AEP6 CDS 3' incomplete TSL:5

Abca5-203 ENSMUST00000127318.7 3580 No protein - Retained intron - - TSL:1

Abca5-205 ENSMUST00000148984.1 2684 No protein - lncRNA - - TSL:1

88.35 kb Forward strand

110.26Mb 110.28Mb 110.30Mb 110.32Mb 110.34Mb Contigs < AL603792.12 AL671964.7 > Genes < Abca5-201protein coding (Comprehensive set...

< Abca5-202protein coding

< Abca5-203retained intron

< Abca5-204protein coding

< Abca5-205lncRNA

Regulatory Build

110.26Mb 110.28Mb 110.30Mb 110.32Mb 110.34Mb Reverse strand 88.35 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000043961

< Abca5-201protein coding

Reverse strand 68.31 kb

ENSMUSP00000047... Transmembrane heli... Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase SMART AAA+ ATPase domain Pfam PF12698 ABC transporter-like PROSITE profiles ABC transporter-like PROSITE patterns ABC transporter, conserved site PANTHER ATP-binding cassette subfamily A member 5

ABC transporter A Gene3D 3.40.50.300 CDD cd03263

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1642

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

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