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Mouse Tlr6 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tlr6 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tlr6 gene (NCBI Reference Sequence: NM_011604 ; Ensembl: ENSMUSG00000051498 ) is located on Mouse chromosome 5. 2 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000062315). 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 Tlr6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-249H4 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: Inactivation of this gene results in abnormal macrophage function.

Exon 2 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 2. The size of effective cKO region: ~2722 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele T gRNA region G 5' A 3'

1 2

Targeting vector T G A

Targeted allele T G A

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tlr6 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(8418bp) | A(27.8% 2340) | C(22.51% 1895) | T(29.25% 2462) | G(20.44% 1721)

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 - 64955563 64958562 3000 browser details YourSeq 72 38 159 3000 86.8% chr16 - 37421120 37421246 127 browser details YourSeq 68 38 267 3000 81.0% chr10 + 95204714 95204931 218 browser details YourSeq 56 38 164 3000 84.2% chr10 - 72598726 72598852 127 browser details YourSeq 56 28 112 3000 79.8% chrX + 9745894 9745968 75 browser details YourSeq 47 38 104 3000 94.4% chr1 - 65614754 65614827 74 browser details YourSeq 44 52 159 3000 92.4% chr2 - 68616855 68616962 108 browser details YourSeq 44 38 162 3000 90.8% chr12 + 46520264 46520392 129 browser details YourSeq 35 83 130 3000 97.3% chr4 - 136967279 136967865 587 browser details YourSeq 34 27 80 3000 92.5% chr3 - 127223105 127223160 56 browser details YourSeq 34 14 63 3000 77.8% chr1 - 21870525 21870571 47 browser details YourSeq 32 103 162 3000 83.0% chr2 - 125446771 125446829 59 browser details YourSeq 30 108 162 3000 94.0% chr2 - 72695602 72695659 58 browser details YourSeq 25 40 65 3000 100.0% chr8 + 103590253 103590280 28 browser details YourSeq 24 1066 1097 3000 87.5% chr1 - 60318691 60318722 32 browser details YourSeq 24 291 320 3000 96.2% chr18 + 43689301 43689332 32 browser details YourSeq 22 1155 1182 3000 89.3% chr3 - 85608070 85608097 28 browser details YourSeq 22 2594 2618 3000 95.9% chr18 - 33724071 33724097 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% chr5 - 64950145 64953144 3000 browser details YourSeq 54 1308 1369 3000 93.6% chr17 + 25288765 25288826 62 browser details YourSeq 53 2553 2899 3000 68.5% chr12 + 73963270 73963410 141 browser details YourSeq 45 1310 1369 3000 88.4% chr11 + 49860254 49860315 62 browser details YourSeq 45 1310 1369 3000 94.2% chr10 + 19431476 19431537 62 browser details YourSeq 44 1310 1369 3000 86.7% chr1 - 104472848 104472907 60 browser details YourSeq 43 2450 2529 3000 80.3% chr10 - 91357353 91357433 81 browser details YourSeq 43 1310 1369 3000 97.8% chr3 + 96460986 96461047 62 browser details YourSeq 41 2559 2606 3000 95.6% chr9 - 113866560 113866608 49 browser details YourSeq 41 1311 1369 3000 93.7% chr7 - 82545221 82545281 61 browser details YourSeq 41 2531 2585 3000 80.0% chr12 - 39235288 39235335 48 browser details YourSeq 41 1310 1369 3000 85.0% chr10 - 128749666 128749727 62 browser details YourSeq 41 1329 1369 3000 100.0% chr18 + 37839197 37839237 41 browser details YourSeq 40 2414 2459 3000 93.5% chr2 - 116944363 116944408 46 browser details YourSeq 38 1332 1369 3000 100.0% chr13 - 93445405 93445442 38 browser details YourSeq 38 1328 1365 3000 100.0% chr12 - 4921905 4921942 38 browser details YourSeq 38 1332 1369 3000 100.0% chr8 + 106712395 106712432 38 browser details YourSeq 38 1332 1369 3000 100.0% chr7 + 27578270 27578307 38 browser details YourSeq 38 1332 1369 3000 100.0% chr2 + 132493570 132493607 38 browser details YourSeq 38 1332 1369 3000 100.0% chr15 + 12508625 12508662 38

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: Tlr6 toll-like receptor 6 [ Mus musculus (house mouse) ] Gene ID: 21899, updated on 24-Sep-2019

Gene summary

Official Symbol Tlr6 provided by MGI Official Full Name toll-like receptor 6 provided by MGI Primary source MGI:MGI:1341296 See related Ensembl:ENSMUSG00000051498 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 Expression Broad expression in spleen adult (RPKM 3.6), colon adult (RPKM 1.8) and 17 other tissues See more Orthologs human all

Genomic context

Location: 5 C3.1; 5 33.54 cM See Tlr6 in Genome Data Viewer Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (64952030..64970942, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (65344334..65351273, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 2 transcripts

Gene: Tlr6 ENSMUSG00000051498

Description toll-like receptor 6 [Source:MGI Symbol;Acc:MGI:1341296] Location Chromosome 5: 64,952,031-64,960,097 reverse strand. GRCm38:CM000998.2 About this gene This gene has 2 transcripts (splice variants), 187 orthologues, 23 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tlr6-201 ENSMUST00000062315.5 3677 806aa ENSMUSP00000062096.4 Protein coding CCDS19303 Q3UV88 TSL:1 GENCODE basic APPRIS P1

Tlr6-202 ENSMUST00000201307.1 446 113aa ENSMUSP00000143865.1 Protein coding - A0A0J9YTV4 CDS 3' incomplete TSL:1

28.07 kb Forward strand 64.95Mb 64.96Mb 64.97Mb Genes Fam114a1-201 >protein coding (Comprehensive set...

Contigs < AC161757.5 Genes (Comprehensive set... < Tlr6-201protein coding

< Tlr6-202protein coding

Regulatory Build

64.95Mb 64.96Mb 64.97Mb Reverse strand 28.07 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

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Transcript: ENSMUST00000062315

< Tlr6-201protein coding

Reverse strand 8.02 kb

ENSMUSP00000062... Transmembrane heli... Superfamily SSF52058 Toll/interleukin-1 receptor homology (TIR) domain superfamily

SMART Leucine-rich repeat, typical subtype Toll/interleukin-1 receptor homology (TIR) domain

Cysteine-rich flanking region, C-terminal Prints PR01537 Pfam Leucine-rich repeat Toll/interleukin-1 receptor homology (TIR) domain

PROSITE profiles Leucine-rich repeat Toll/interleukin-1 receptor homology (TIR) domain

PIRSF PIRSF037595 PANTHER Toll-like receptor 6

PTHR24365 Gene3D Leucine-rich repeat domain superfamily Toll/interleukin-1 receptor homology (TIR) domain superfamily

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

Variant Legend

missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 806

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

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