https://www.alphaknockout.com Mouse Fgl1 Knockout Project (CRISPR/Cas9) Objective: To create a Fgl1 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fgl1 gene (NCBI Reference Sequence: NM_145594 ; Ensembl: ENSMUSG00000031594 ) is located on Mouse chromosome 8. 8 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000034003). Exon 3~4 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: Mice homozygous for one null allele exhibit increased body weight, white fat and gluconeogenesis, decreased circulating cholesterol, free fatty acid level and respiratory quotient, hyperglycemia, and impaired glucose tolerance. Mice homozygous for a second null allele display normal appearance with age-related onset of dermatitis. Exon 3 starts from about 6.79% of the coding region. Exon 3~4 covers 36.84% of the coding region. The size of effective KO region: ~9439 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 8 Legends Exon of mouse Fgl1 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 543 bp section upstream of Exon 3 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 695 bp section downstream of Exon 4 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(543bp) | A(33.7% 183) | C(17.86% 97) | T(29.47% 160) | G(18.97% 103) Note: The 543 bp section upstream of Exon 3 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(695bp) | A(22.3% 155) | C(17.41% 121) | T(38.13% 265) | G(22.16% 154) Note: The 695 bp section downstream of Exon 4 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 543 1 543 543 100.0% chr8 - 41209884 41210426 543 browser details YourSeq 36 39 76 543 100.0% chr11 + 55458536 55770756 312221 browser details YourSeq 28 201 229 543 100.0% chr15 - 38662546 38662575 30 browser details YourSeq 25 155 180 543 100.0% chr19 - 36841069 36841097 29 browser details YourSeq 25 22 47 543 100.0% chr17 + 28478295 28478323 29 browser details YourSeq 24 136 162 543 96.3% chr1 - 134696895 134696922 28 browser details YourSeq 24 41 67 543 96.3% chr2 + 8425896 8425925 30 browser details YourSeq 23 41 64 543 100.0% chr2 - 84108928 84108956 29 browser details YourSeq 23 169 195 543 92.6% chr2 + 32275926 32275952 27 browser details YourSeq 21 181 203 543 95.7% chr2 - 29096900 29096922 23 browser details YourSeq 21 174 194 543 100.0% chr12 - 5458196 5458216 21 browser details YourSeq 20 41 60 543 100.0% chr11 + 64184901 64184920 20 browser details YourSeq 20 53 72 543 100.0% chr10 + 20417547 20417566 20 Note: The 543 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 695 1 695 695 100.0% chr8 - 41199750 41200444 695 browser details YourSeq 235 86 500 695 87.5% chr4 + 129411747 129412289 543 browser details YourSeq 227 87 500 695 86.5% chrX + 101498396 101499015 620 browser details YourSeq 216 78 500 695 85.7% chr7 - 13059392 13060001 610 browser details YourSeq 215 125 500 695 82.9% chr17 + 28526319 28526647 329 browser details YourSeq 203 67 500 695 81.9% chr5 + 65397076 65397472 397 browser details YourSeq 196 132 500 695 86.7% chr5 + 34391098 34671465 280368 browser details YourSeq 194 138 487 695 87.4% chr3 - 58579777 58580410 634 browser details YourSeq 187 78 500 695 82.2% chr9 + 66602571 66602955 385 browser details YourSeq 179 188 500 695 86.8% chr10 + 40407200 40407483 284 browser details YourSeq 171 115 500 695 84.9% chr6 + 148987454 148987813 360 browser details YourSeq 166 74 500 695 90.7% chr4 + 45340042 45340539 498 browser details YourSeq 159 188 500 695 90.0% chr17 - 24458495 24807709 349215 browser details YourSeq 154 110 514 695 89.3% chr17 - 83612055 83612643 589 browser details YourSeq 152 336 514 695 92.8% chr7 + 80289410 80289591 182 browser details YourSeq 150 202 500 695 91.8% chr17 - 85054234 85054574 341 browser details YourSeq 148 46 500 695 88.3% chr7 + 127639949 127640482 534 browser details YourSeq 146 336 531 695 89.3% chr12 + 59167277 59167473 197 browser details YourSeq 141 235 492 695 92.3% chr3 - 58365711 58366061 351 browser details YourSeq 141 339 510 695 91.3% chr11 - 26541347 26541709 363 Note: The 695 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Fgl1 fibrinogen-like protein 1 [ Mus musculus (house mouse) ] Gene ID: 234199, updated on 12-Aug-2019 Gene summary Official Symbol Fgl1 provided by MGI Official Full Name fibrinogen-like protein 1 provided by MGI Primary source MGI:MGI:102795 See related Ensembl:ENSMUSG00000031594 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 Mfire1 Expression Restricted expression toward liver E18 (RPKM 174.3) See more Orthologs human all Genomic context Location: 8; 8 A4 See Fgl1 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (41191434..41217978, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (42276788..42300510, complement) Chromosome 8 - NC_000074.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Fgl1 ENSMUSG00000031594 Description fibrinogen-like protein 1 [Source:MGI Symbol;Acc:MGI:102795] Location Chromosome 8: 41,191,434-41,215,156 reverse strand. GRCm38:CM001001.2 About this gene This gene has 2 transcripts (splice variants), 130 orthologues, 23 paralogues, is a member of 1 Ensembl protein family and is associated with 26 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fgl1-201 ENSMUST00000034003.4 1120 314aa ENSMUSP00000034003.4 Protein coding CCDS22260 A0A0R4J0E1 TSL:1 GENCODE basic APPRIS P1 Fgl1-202 ENSMUST00000134510.1 3907 No protein - Retained intron - - TSL:2 43.72 kb Forward strand 41.19Mb 41.20Mb 41.21Mb 41.22Mb Genes Gm16348-202 >lncRNA (Comprehensive set... Gm16348-201 >lncRNA Contigs < AC156554.13 Genes (Comprehensive set... < Fgl1-201protein coding < Fgl1-202retained intron Regulatory Build 41.19Mb 41.20Mb 41.21Mb 41.22Mb Reverse strand 43.72 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000034003 < Fgl1-201protein coding Reverse strand 23.72 kb ENSMUSP00000034... Coiled-coils (Ncoils) Cleavage site (Sign... Superfamily Fibrinogen-like, C-terminal SMART Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain Pfam Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain PROSITE profiles Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain PROSITE patterns Fibrinogen, conserved site PANTHER PTHR19143 PTHR19143:SF263 Gene3D 3.90.215.20 CDD Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 314 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.