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Mouse Tom1l1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tom1l1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tom1l1 gene (NCBI Reference Sequence: NM_028011 ; Ensembl: ENSMUSG00000020541 ) is located on Mouse chromosome 11. 16 exons are identified, with the ATG start codon in exon 4 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000107868). Exon 9~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tom1l1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-71O2 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 9 starts from about 52.14% of the coding region. The knockout of Exon 9~10 will result in frameshift of the gene. The size of intron 8 for 5'-loxP site insertion: 3547 bp, and the size of intron 10 for 3'-loxP site insertion: 1263 bp. The size of effective cKO region: ~995 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 9 10 11 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tom1l1 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(7495bp) | A(28.7% 2151) | C(20.39% 1528) | T(30.37% 2276) | G(20.55% 1540)

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 - 90658459 90661458 3000 browser details YourSeq 175 2249 2678 3000 87.1% chr16 + 95647252 95647494 243 browser details YourSeq 165 2499 2678 3000 97.2% chr14 - 56662553 56662732 180 browser details YourSeq 165 2294 2678 3000 92.3% chr12 - 90514880 90515258 379 browser details YourSeq 161 2497 2690 3000 92.1% chr14 - 47450978 47451164 187 browser details YourSeq 159 2498 2678 3000 94.2% chr19 - 23098224 23098401 178 browser details YourSeq 159 2499 2680 3000 96.0% chr16 + 64793018 64793202 185 browser details YourSeq 158 2517 2709 3000 89.8% chr15 - 26740057 26740231 175 browser details YourSeq 157 2509 2678 3000 95.9% chr18 - 77628962 77629130 169 browser details YourSeq 157 2520 2712 3000 90.3% chr7 + 45658871 45659051 181 browser details YourSeq 157 2517 2680 3000 98.2% chr19 + 34823131 34823348 218 browser details YourSeq 156 2517 2678 3000 98.2% chr15 + 59660697 59660858 162 browser details YourSeq 155 2482 2667 3000 96.5% chr5 + 147255649 147255840 192 browser details YourSeq 154 2506 2706 3000 87.5% chr2 + 168215534 168215710 177 browser details YourSeq 153 2520 2678 3000 96.9% chr17 - 64373778 64373935 158 browser details YourSeq 153 2516 2678 3000 97.0% chr9 + 21486414 21486576 163 browser details YourSeq 153 2517 2705 3000 90.1% chr3 + 58580283 58580455 173 browser details YourSeq 153 2517 2678 3000 98.2% chr11 + 21074631 21074792 162 browser details YourSeq 152 2517 2678 3000 97.0% chr1 + 13075727 13075888 162 browser details YourSeq 151 2499 2678 3000 89.6% chr4 - 149776525 149776696 172

Note: The 3000 bp section upstream of Exon 9 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 - 90654464 90657463 3000 browser details YourSeq 141 1238 1731 3000 90.3% chr16 + 30635919 30636422 504 browser details YourSeq 138 1328 1807 3000 80.3% chr1 + 61127721 61128206 486 browser details YourSeq 137 1239 1683 3000 85.2% chr2 - 127549701 127550143 443 browser details YourSeq 137 1242 1731 3000 85.2% chr18 - 66481548 66482031 484 browser details YourSeq 136 1238 1731 3000 75.8% chr2 + 129861509 129861964 456 browser details YourSeq 134 1245 1683 3000 76.7% chr7 - 132450828 132451240 413 browser details YourSeq 134 1238 1683 3000 78.0% chr2 + 20976322 20976732 411 browser details YourSeq 126 1243 1683 3000 75.0% chr2 - 25590537 25590848 312 browser details YourSeq 125 1249 1701 3000 83.6% chr4 + 81051152 81051594 443 browser details YourSeq 123 1238 1680 3000 81.1% chr16 - 91205517 91205916 400 browser details YourSeq 123 1258 1729 3000 80.3% chr1 - 151147789 151148239 451 browser details YourSeq 121 1179 1683 3000 79.1% chrX - 153619775 153620260 486 browser details YourSeq 120 1162 1683 3000 83.9% chr15 - 12685258 12685848 591 browser details YourSeq 120 1329 1774 3000 91.1% chr5 + 137991364 137991986 623 browser details YourSeq 119 1235 1683 3000 81.1% chr6 - 49138166 49138592 427 browser details YourSeq 118 1235 1679 3000 80.8% chr11 + 19893418 19893782 365 browser details YourSeq 115 1237 1674 3000 78.5% chr9 + 120401193 120401552 360 browser details YourSeq 114 1238 1654 3000 88.2% chr12 + 39860634 39861050 417 browser details YourSeq 114 1190 1683 3000 81.5% chr12 + 17399553 17399982 430

Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tom1l1 target of myb1-like 1 (chicken) [ Mus musculus (house mouse) ] Gene ID: 71943, updated on 24-Oct-2019

Gene summary

Official Symbol Tom1l1 provided by MGI Official Full Name target of myb1-like 1 (chicken) provided by MGI Primary source MGI:MGI:1919193 See related Ensembl:ENSMUSG00000020541 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 C80573; Srcasm; 2310045L10Rik Expression Ubiquitous expression in liver E14 (RPKM 14.0), liver E14.5 (RPKM 12.2) and 28 other tissues See more Orthologs human all

Genomic context

Location: 11 D; 11 55.47 cM See Tom1l1 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (90645690..90688279, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (90507005..90548915, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 8 transcripts

Gene: Tom1l1 ENSMUSG00000020541

Description target of myb1-like 1 (chicken) [Source:MGI Symbol;Acc:MGI:1919193] Gene Synonyms 2310045L10Rik, Srcasm Location Chromosome 11: 90,643,465-90,688,366 reverse strand. GRCm38:CM001004.2 About this gene This gene has 8 transcripts (splice variants), 158 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tom1l1-203 ENSMUST00000107868.7 2093 397aa ENSMUSP00000103500.1 Protein coding CCDS36280 Q8BZR6 TSL:1 GENCODE basic

Tom1l1-201 ENSMUST00000020849.8 4308 474aa ENSMUSP00000020849.2 Protein coding - Q923U0 TSL:1 GENCODE basic APPRIS P1

Tom1l1-202 ENSMUST00000107867.7 2439 227aa ENSMUSP00000103499.1 Protein coding - Q923U0 TSL:1 GENCODE basic

Tom1l1-204 ENSMUST00000107869.8 1826 398aa ENSMUSP00000103501.2 Protein coding - Q923U0 TSL:1 GENCODE basic

Tom1l1-208 ENSMUST00000154599.1 592 191aa ENSMUSP00000123329.1 Protein coding - A6PWP3 CDS 3' incomplete TSL:3

Tom1l1-207 ENSMUST00000147329.1 2694 No protein - Retained intron - - TSL:2

Tom1l1-205 ENSMUST00000127034.1 728 No protein - Retained intron - - TSL:5

Tom1l1-206 ENSMUST00000131055.1 749 No protein - lncRNA - - TSL:3

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64.90 kb Forward strand 90.64Mb 90.65Mb 90.66Mb 90.67Mb 90.68Mb 90.69Mb Genes Cox11-201 >protein coding (Comprehensive set...

Cox11-202 >protein coding

Contigs AL646046.8 > AL672199.16 > Genes (Comprehensive set... < Stxbp4-207protein coding < Tom1l1-201protein coding

< Stxbp4-201protein coding < Tom1l1-202protein coding < Tom1l1-208protein coding

< Stxbp4-203protein coding < Tom1l1-203protein coding

< Stxbp4-202protein coding < Tom1l1-204protein coding

< Stxbp4-205lncRNA < Tom1l1-205retained intron < Tom1l1-207retained intron < Tom1l1-206lncRNA

Regulatory Build

90.64Mb 90.65Mb 90.66Mb 90.67Mb 90.68Mb 90.69Mb Reverse strand 64.90 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000107868

< Tom1l1-203protein coding

Reverse strand 41.91 kb

ENSMUSP00000103... MobiDB lite Low complexity (Seg) Superfamily ENTH/VHS SSF89009

Pfam VHS domain GAT domain

PROSITE profiles VHS domain GAT domain

PIRSF Target of Myb protein 1 PANTHER TOM1-like protein 1

PTHR13856 Gene3D ENTH/VHS GAT domain superfamily

CDD cd16997 cd14237

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 397

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

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