http://www.alphaknockout.com/ Mouse Slc2a6 Knockout Project (CRISPR/Cas9)

Objective: To create a Slc2a6 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Slc2a6 (NCBI Reference Sequence: NM_172659 ; Ensembl: ENSMUSG00000036067 ) is located on Mouse 2. 10 exons are identified , with the ATG start codon in exon 1 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000045702). Exon 2 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: Homozygous null females fed a Western diet exhibit decreased adiposity and females fed a chow diet, but not Western diet, show lower respiratory exchange ratio.

Exon 2 starts from about 6.04% of the coding region. Exon 2 covers 10.93% of the coding region. The size of effective KO region: ~960 bp. The KO 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 10

Legends Exon of mouse Slc2a6 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 513 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1033 bp section downstream of Exon 2 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(513bp) | A(24.17% 124) | C(26.9% 138) | G(27.29% 140) | T(21.64% 111)

Note: The 513 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(1033bp) | A(25.85% 267) | C(26.04% 269) | G(25.56% 264) | T(22.56% 233)

Note: The 1033 bp section downstream 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 513 1 513 513 100.0% chr2 - 27027304 27027816 513 browser details YourSeq 29 339 376 513 77.5% chr16 - 47231340 47231371 32 browser details YourSeq 23 10 35 513 83.4% chr15 + 12775150 12775173 24 browser details YourSeq 22 339 362 513 87.0% chr11 - 22830754 22830776 23 browser details YourSeq 21 345 365 513 100.0% chr2 + 157563706 157563726 21 browser details YourSeq 20 442 461 513 100.0% chr10 - 77511541 77511560 20 browser details YourSeq 20 463 482 513 100.0% chr17 + 22408481 22408500 20 browser details YourSeq 20 102 121 513 100.0% chr16 + 60264591 60264610 20 browser details YourSeq 20 446 465 513 100.0% chr14 + 20450081 20450100 20

Note: The 513 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 1033 1 1033 1033 100.0% chr2 - 27026108 27027140 1033 browser details YourSeq 37 344 760 1033 68.3% chr1 - 182044383 182044757 375 browser details YourSeq 34 732 775 1033 83.8% chr12 - 52763972 52764014 43 browser details YourSeq 34 722 759 1033 94.8% chr5 + 140356712 140356749 38 browser details YourSeq 31 732 776 1033 94.3% chrX - 162714394 162714441 48 browser details YourSeq 30 718 747 1033 100.0% chr17 - 34865417 34865446 30 browser details YourSeq 28 731 762 1033 93.8% chr14 - 75024736 75024767 32 browser details YourSeq 27 733 761 1033 96.6% chr11 + 29557015 29557043 29 browser details YourSeq 26 713 740 1033 96.5% chr19 - 21615615 21615642 28 browser details YourSeq 25 731 757 1033 96.3% chr3 - 88218285 88218311 27 browser details YourSeq 25 19 47 1033 93.2% chr2 + 131093301 131093329 29 browser details YourSeq 25 721 747 1033 96.3% chr19 + 60788001 60788027 27 browser details YourSeq 25 723 757 1033 75.0% chr18 + 38616639 38616670 32 browser details YourSeq 25 720 752 1033 73.1% chr1 + 53597941 53597966 26 browser details YourSeq 24 732 757 1033 96.2% chr4 - 33047321 33047346 26 browser details YourSeq 24 732 757 1033 96.2% chr2 + 167821721 167821746 26 browser details YourSeq 22 725 748 1033 95.9% chr16 - 32382239 32382262 24 browser details YourSeq 22 739 760 1033 100.0% chr10 - 128986907 128986928 22 browser details YourSeq 22 736 757 1033 100.0% chr1 + 157241660 157241681 22 browser details YourSeq 22 720 747 1033 89.3% chr1 + 68662737 68662764 28

Note: The 1033 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 http://www.alphaknockout.com/ Gene and information: Slc2a6 2 (facilitated ), member 6 [ Mus musculus (house mouse) ] Gene ID: 227659, updated on 26-Jun-2020

Gene summary

Official Symbol Slc2a6 provided by MGI Official Full Name solute carrier family 2 (facilitated glucose transporter), member 6 provided by MGI Primary source MGI:MGI:2443286 See related Ensembl:ENSMUSG00000036067 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 Glut6; Glut9; GLUT-6; A330096C23; F630103L12Rik Expression Broad expression in cerebellum adult (RPKM 12.1), spleen adult (RPKM 11.0) and 20 other tissuesS ee more Orthologs human all

Genomic context

Location: 2; 2 A3 See Slc2a6 in Genome Data Viewer Exon count: 10

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (27021363..27028000, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (26876885..26883518, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 6 transcripts

Gene: Slc2a6 ENSMUSG00000036067

Description solute carrier family 2 (facilitated glucose transporter), member 6 [Source:MGI Symbol;Acc:MGI:2443286] Gene Synonyms F630103L12Rik, Glut6 Location Chromosome 2: 27,021,363-27,027,998 reverse strand. GRCm38:CM000995.2 About this gene This gene has 6 transcripts (splice variants), 347 orthologues, 11 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Slc2a6-201 ENSMUST00000045702.5 2138 497aa ENSMUSP00000049103.5 Protein coding CCDS15822 Q3UDF0 TSL:1 GENCODE basic APPRIS P3

Slc2a6-202 ENSMUST00000102890.10 1887 443aa ENSMUSP00000099954.4 Protein coding CCDS50544 Q3UDF0 TSL:1 GENCODE basic APPRIS ALT2

Slc2a6-206 ENSMUST00000153388.1 343 85aa ENSMUSP00000122054.1 Protein coding - A2AR27 CDS 3' incomplete TSL:5

Slc2a6-203 ENSMUST00000129835.7 2711 No protein - Processed transcript - - TSL:1

Slc2a6-205 ENSMUST00000145742.7 2347 No protein - Processed transcript - - TSL:1

Slc2a6-204 ENSMUST00000135725.1 668 No protein - Processed transcript - - TSL:3

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26.64 kb Forward strand 27.015Mb 27.020Mb 27.025Mb 27.030Mb 27.035Mb Cacfd1-205 >protein coding Gm13397-201 >processed pseudogene (Comprehensive set...

Cacfd1-204 >protein coding Gm13398-201 >protein coding

Cacfd1-202 >protein coding

Cacfd1-203 >protein coding

Cacfd1-206 >processed transcript

Cacfd1-207 >protein coding

Cacfd1-201 >protein coding

Cacfd1-208 >retained intron

Contigs AL845266.2 > Genes < Slc2a6-201protein coding (Comprehensive set...

< Slc2a6-203processed transcript

< Slc2a6-205processed transcript

< Slc2a6-202protein coding

< Slc2a6-204processed transcript

< Slc2a6-206protein coding

Regulatory Build

27.015Mb 27.020Mb 27.025Mb 27.030Mb 27.035Mb Reverse strand 26.64 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

pseudogene processed transcript

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Transcript: ENSMUST00000045702

< Slc2a6-201protein coding

Reverse strand 6.64 kb

ENSMUSP00000049... Transmembrane heli... Low complexity (Seg) TIGRFAM Sugar/inositol transporter Superfamily MFS transporter superfamily Prints Sugar/inositol transporter Pfam Major facilitator, sugar transporter-like PROSITE profiles Major facilitator superfamily domain PROSITE patterns Sugar transporter, conserved site Sugar transporter, conserved site

PANTHER PTHR23500:SF111

PTHR23500 Gene3D 1.20.1250.20 CDD cd17434

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 497

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

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