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Mouse Slc46a3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Slc46a3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Slc46a3 (NCBI Reference Sequence: NM_027872 ; Ensembl: ENSMUSG00000029650 ) is located on Mouse 5. 6 are identified, with the ATG in 2 and the TAG stop codon in exon 6 (Transcript: ENSMUST00000031655). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Slc46a3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-223M24 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 3 starts from about 13.77% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of 2 for 5'-loxP site insertion: 6991 bp, and the size of intron 3 for 3'-loxP site insertion: 1690 bp. The size of effective cKO region: ~1371 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 3 4 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Slc46a3 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7871bp) | A(27.14% 2136) | C(22.09% 1739) | T(27.65% 2176) | G(23.12% 1820)

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% chr5 - 147887092 147890091 3000 browser details YourSeq 264 2460 2779 3000 91.8% chr8 - 120386031 120386528 498 browser details YourSeq 263 2455 2780 3000 90.6% chr12 + 111492026 111492350 325 browser details YourSeq 260 2460 2780 3000 90.0% chr14 - 65160225 65160543 319 browser details YourSeq 257 2460 2780 3000 88.9% chr7 - 24121662 24121976 315 browser details YourSeq 256 2475 2780 3000 92.4% chr5 - 116666396 116666703 308 browser details YourSeq 256 2466 2780 3000 89.6% chr12 - 80703212 80703520 309 browser details YourSeq 255 2461 2780 3000 89.2% chr2 - 168357463 168357777 315 browser details YourSeq 255 2466 2781 3000 91.1% chr8 + 11456569 11456902 334 browser details YourSeq 254 2044 2780 3000 84.2% chr5 + 116764085 116764422 338 browser details YourSeq 254 2460 2780 3000 89.0% chr10 + 63180574 63180892 319 browser details YourSeq 252 2460 2781 3000 92.0% chr7 - 24266756 24267094 339 browser details YourSeq 252 2452 2779 3000 87.4% chr14 - 118829985 118830308 324 browser details YourSeq 252 2457 2780 3000 87.7% chrX + 94336570 94336885 316 browser details YourSeq 252 2447 2780 3000 87.9% chr1 + 39404520 39404840 321 browser details YourSeq 251 2460 2780 3000 89.8% chr6 + 143424564 143424883 320 browser details YourSeq 251 2466 2780 3000 90.4% chr2 + 167204829 167205161 333 browser details YourSeq 251 2460 2781 3000 88.4% chr17 + 15339429 15339747 319 browser details YourSeq 251 2453 2781 3000 87.4% chr15 + 100927764 100928089 326 browser details YourSeq 250 2457 2778 3000 90.1% chr7 - 35624973 35625309 337

Note: The 3000 bp section upstream of Exon 3 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% chr5 - 147882721 147885720 3000 browser details YourSeq 252 479 2851 3000 92.4% chr1 - 4833480 4912997 79518 browser details YourSeq 151 2674 2848 3000 97.5% chr9 - 43136394 43136681 288 browser details YourSeq 148 2697 2868 3000 94.5% chr1 + 87682431 87682601 171 browser details YourSeq 145 2670 2851 3000 94.0% chr6 + 24359156 24359486 331 browser details YourSeq 145 2698 2851 3000 97.4% chr11 + 74358663 74358820 158 browser details YourSeq 144 2697 2853 3000 96.2% chr2 - 146756060 146756218 159 browser details YourSeq 144 2696 2851 3000 96.8% chr11 - 98830686 98831022 337 browser details YourSeq 144 2697 2851 3000 96.8% chr1 - 60069574 60069764 191 browser details YourSeq 144 2694 2848 3000 96.8% chr9 + 110106802 110107023 222 browser details YourSeq 143 2698 2852 3000 96.2% chr5 + 119653422 119653576 155 browser details YourSeq 142 2694 2851 3000 95.6% chr1 - 74241756 74241924 169 browser details YourSeq 141 2699 2848 3000 97.4% chr9 - 73079604 73079780 177 browser details YourSeq 141 2695 2844 3000 97.4% chr18 + 66873996 66874147 152 browser details YourSeq 140 2698 2848 3000 96.7% chr6 - 148893334 148893488 155 browser details YourSeq 140 2698 2848 3000 96.0% chr1 + 89062175 89062324 150 browser details YourSeq 139 2698 2847 3000 96.7% chr8 - 112169180 112169339 160 browser details YourSeq 139 2695 2851 3000 95.0% chr5 - 34668710 34668868 159 browser details YourSeq 139 2698 2851 3000 96.1% chr19 - 37454636 37454791 156 browser details YourSeq 139 2698 2851 3000 94.8% chr18 - 57383611 57383763 153

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 information: Slc46a3 46, member 3 [ Mus musculus () ] Gene ID: 71706, updated on 24-Oct-2019

Gene summary

Official Symbol Slc46a3 provided by MGI Official Full Name solute carrier family 46, member 3 provided by MGI Primary source MGI:MGI:1918956 See related Ensembl:ENSMUSG00000029650 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 1200006F02Rik Expression Broad expression in large intestine adult (RPKM 23.2), adult (RPKM 22.0) and 23 other tissues See more Orthologs all

Genomic context

Location: 5; 5 G3 See Slc46a3 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (147875897..147894861, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (148690017..148706378, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 4 transcripts

Gene: Slc46a3 ENSMUSG00000029650

Description solute carrier family 46, member 3 [Source:MGI Symbol;Acc:MGI:1918956] Gene Synonyms 1200006F02Rik Location Chromosome 5: 147,878,437-147,894,815 reverse strand. GRCm38:CM000998.2 About this gene This gene has 4 transcripts (splice variants), 218 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein ID Biotype CCDS UniProt Flags

Slc46a3-201 ENSMUST00000031655.3 2434 460aa ENSMUSP00000031655.3 Protein coding CCDS19880 Q9DC26 TSL:1 GENCODE basic APPRIS P1

Slc46a3-202 ENSMUST00000118527.7 2210 460aa ENSMUSP00000113879.1 Protein coding CCDS19880 Q9DC26 TSL:1 GENCODE basic APPRIS P1

Slc46a3-203 ENSMUST00000138244.1 388 47aa ENSMUSP00000120032.1 Protein coding - D3YWM2 CDS 3' incomplete TSL:2

Slc46a3-204 ENSMUST00000152064.1 833 No protein - Retained intron - - TSL:2

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36.38 kb Forward strand 147.87Mb 147.88Mb 147.89Mb 147.90Mb Pomp-201 >protein coding (Comprehensive set...

Pomp-203 >protein coding

Pomp-202 >protein coding

Pomp-204 >lncRNA

Pomp-206 >retained intron

Contigs AC122299.5 > < AC133954.3 Genes (Comprehensive set... < Gm35648-201processed pseudogene < Slc46a3-202protein coding

< Slc46a3-201protein coding

< Slc46a3-204retained intron < Slc46a3-203protein coding

Regulatory Build

147.87Mb 147.88Mb 147.89Mb 147.90Mb Reverse strand 36.38 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Flank Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene pseudogene

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Transcript: ENSMUST00000031655

< Slc46a3-201protein coding

Reverse strand 16.38 kb

ENSMUSP00000031... Transmembrane heli... Low complexity (Seg) Superfamily MFS transporter superfamily Pfam Major facilitator superfamily PANTHER PTHR23507

PTHR23507:SF9 Gene3D 1.20.1250.20 CDD cd17448

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 460

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

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