https://www.alphaknockout.com Mouse Frem1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Frem1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Frem1 gene (NCBI Reference Sequence: NM_001198811 ; Ensembl: ENSMUSG00000059049 ) is located on Mouse chromosome 4. 37 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 37 (Transcript: ENSMUST00000107230). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Frem1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-91B7 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: Homozygous mutation of this gene results in subepidermal blistering, cryptophthalmos, syndactyly, and renal agenesis. Exon 4 starts from about 5.34% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2260 bp, and the size of intron 4 for 3'-loxP site insertion: 1755 bp. The size of effective cKO region: ~802 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 5 37 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Frem1 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com 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(7302bp) | A(28.77% 2101) | C(21.02% 1535) | T(28.61% 2089) | G(21.6% 1577) 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. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr4 - 83014366 83017365 3000 browser details YourSeq 96 200 662 3000 87.5% chr13 + 98962845 98963707 863 browser details YourSeq 91 205 505 3000 89.6% chrX - 84423691 84424020 330 browser details YourSeq 90 332 664 3000 93.3% chr4 + 142406488 142406832 345 browser details YourSeq 86 205 711 3000 87.8% chr1 + 164526443 164527171 729 browser details YourSeq 80 477 645 3000 84.5% chr10 - 42975779 42975955 177 browser details YourSeq 78 205 562 3000 69.8% chr7 + 127161103 127161440 338 browser details YourSeq 75 371 645 3000 72.8% chr18 - 11108830 11109077 248 browser details YourSeq 74 495 679 3000 72.1% chr4 - 104131013 104131200 188 browser details YourSeq 74 490 644 3000 80.0% chr6 + 88723719 88723916 198 browser details YourSeq 73 205 515 3000 92.9% chr7 - 65827314 65827635 322 browser details YourSeq 62 333 542 3000 75.0% chr4 + 146256228 146256356 129 browser details YourSeq 62 333 542 3000 75.0% chr4 + 145767343 145767471 129 browser details YourSeq 59 525 662 3000 76.8% chr14 - 73734408 73734545 138 browser details YourSeq 58 379 645 3000 81.1% chr16 - 92892842 92893104 263 browser details YourSeq 53 531 642 3000 74.8% chrX - 169463200 169463312 113 browser details YourSeq 52 461 568 3000 84.3% chr13 - 96888775 96888882 108 browser details YourSeq 50 459 545 3000 87.0% chr6 - 82663620 82663707 88 browser details YourSeq 47 699 779 3000 79.1% chr1 - 74831181 74831261 81 browser details YourSeq 47 371 542 3000 71.0% chr6 + 58592781 58592872 92 Note: The 3000 bp section upstream of Exon 4 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% chr4 - 83010564 83013563 3000 browser details YourSeq 287 1861 2363 3000 83.0% chr1 + 153778068 153778527 460 browser details YourSeq 249 1851 2327 3000 79.7% chr10 - 18822545 18822987 443 browser details YourSeq 232 1910 2374 3000 87.5% chr16 - 15403206 15403718 513 browser details YourSeq 232 1974 2367 3000 85.1% chr12 + 15069597 15070007 411 browser details YourSeq 220 1974 2357 3000 85.0% chr13 - 30951371 30951836 466 browser details YourSeq 214 1968 2373 3000 85.0% chr15 - 56054667 56055056 390 browser details YourSeq 214 1966 2362 3000 86.8% chr9 + 75467055 75467444 390 browser details YourSeq 184 2045 2363 3000 86.0% chr19 - 51815739 51816053 315 browser details YourSeq 180 1966 2374 3000 88.6% chr7 - 54678869 54679285 417 browser details YourSeq 175 2036 2373 3000 87.3% chr8 + 80665098 80665431 334 browser details YourSeq 158 2036 2374 3000 88.2% chr4 - 22717967 22718445 479 browser details YourSeq 149 819 1335 3000 82.3% chr14 + 76451542 76451934 393 browser details YourSeq 147 817 991 3000 93.4% chr6 - 125395581 125395754 174 browser details YourSeq 145 814 991 3000 92.4% chr1 - 44764444 44764621 178 browser details YourSeq 144 818 992 3000 92.9% chr2 - 120561430 120561632 203 browser details YourSeq 143 819 993 3000 90.6% chr11 - 106209866 106210030 165 browser details YourSeq 143 817 988 3000 90.7% chr14 + 74393333 74393497 165 browser details YourSeq 141 819 999 3000 88.5% chr1 - 85101380 85101552 173 browser details YourSeq 139 819 988 3000 90.8% chr16 - 18569444 18569610 167 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Frem1 Fras1 related extracellular matrix protein 1 [ Mus musculus (house mouse) ] Gene ID: 329872, updated on 12-Aug-2019 Gene summary Official Symbol Frem1 provided by MGI Official Full Name Fras1 related extracellular matrix protein 1 provided by MGI Primary source MGI:MGI:2670972 See related Ensembl:ENSMUSG00000059049 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 heb; crf11; eyes2; qbrick; BC037594; D430009N09; D630008K06; eye<m02Jus> Expression Broad expression in limb E14.5 (RPKM 2.7), CNS E11.5 (RPKM 2.1) and 19 other tissues See more Orthologs human all Genomic context Location: 4; 4 C3 See Frem1 in Genome Data Viewer Exon count: 41 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (82897920..83052506, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (82543831..82667006, complement) Chromosome 4 - NC_000070.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Frem1 ENSMUSG00000059049 Description Fras1 related extracellular matrix protein 1 [Source:MGI Symbol;Acc:MGI:2670972] Gene Synonyms QBRICK, crf11, eye, eyes2, heb Location Chromosome 4: 82,897,920-83,052,339 reverse strand. GRCm38:CM000997.2 About this gene This gene has 8 transcripts (splice variants), 201 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 49 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Frem1- ENSMUST00000107230.7 9690 2172aa ENSMUSP00000102849.1 Protein coding CCDS57281 A2ADN1 TSL:5 202 Q684R7 GENCODE basic APPRIS ALT2 Frem1- ENSMUST00000071708.11 9307 2191aa ENSMUSP00000071627.5 Protein coding CCDS38791 Q684R7 TSL:1 201 GENCODE basic APPRIS P3 Frem1- ENSMUST00000170248.8 9527 2173aa ENSMUSP00000125809.2 Protein coding - E9Q5Z7 TSL:5 208 GENCODE basic APPRIS ALT2 Frem1- ENSMUST00000127886.1 3574 415aa ENSMUSP00000122467.1 Protein coding - F7D5I3 CDS 5' 203 incomplete TSL:1 Frem1- ENSMUST00000130889.1 1252 No - Retained - - TSL:1 204 protein intron Frem1- ENSMUST00000141841.1 632 No - Retained - - TSL:5 207 protein intron Frem1- ENSMUST00000131102.1 3891 No - lncRNA - - TSL:5 205 protein Frem1- ENSMUST00000133610.1 634 No - lncRNA - - TSL:1 206 protein Page 6 of 8 https://www.alphaknockout.com 174.42 kb Forward strand 82.90Mb 82.95Mb 83.00Mb 83.05Mb Contigs AL670958.4 > Genes (Comprehensive set... < Frem1-208protein coding < Frem1-201protein coding < Frem1-202protein coding < Frem1-206lncRNA < Frem1-207retained intron < Frem1-205lncRNA < Frem1-204retained intron < Frem1-203protein coding Regulatory Build 82.90Mb 82.95Mb 83.00Mb 83.05Mb Reverse strand 174.42 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000107230 < Frem1-202protein coding Reverse strand 154.41 kb ENSMUSP00000102... Low complexity (Seg) Cleavage site (Sign... Superfamily C-type lectin fold CalX-like domain superfamily SMART C-type lectin-like Pfam PF16184 Na-Ca exchanger/integrin-beta4 C-type lectin-like PROSITE profiles CSPG repeat C-type lectin-like PANTHER PTHR45739 PTHR45739:SF7 Gene3D CalX-like domain superfamily C-type lectin-like/link domain superfamily CDD cd00037 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2172 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.