https://www.alphaknockout.com Mouse Lyve1 Knockout Project (CRISPR/Cas9) Objective: To create a Lyve1 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Lyve1 gene (NCBI Reference Sequence: NM_053247 ; Ensembl: ENSMUSG00000030787 ) is located on Mouse chromosome 7. 6 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 6 (Transcript: ENSMUST00000033050). Exon 2~5 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 allele display enlarged lymphatic vessels and increased interstitial-lymphatic flow. However, mice homozygous for a second allele do not display any abnormalities in the lymphatic system. Exon 2 starts from about 9.01% of the coding region. Exon 2~5 covers 71.8% of the coding region. The size of effective KO region: ~6930 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 2 3 4 5 6 Legends Exon of mouse Lyve1 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 2000 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 470 bp section downstream of Exon 5 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(2000bp) | A(29.35% 587) | C(22.5% 450) | T(26.55% 531) | G(21.6% 432) Note: The 2000 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(470bp) | A(26.17% 123) | C(19.79% 93) | T(29.57% 139) | G(24.47% 115) Note: The 470 bp section downstream of Exon 5 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 2000 1 2000 2000 100.0% chr7 - 110859759 110861758 2000 browser details YourSeq 56 1 186 2000 83.4% chr5 - 128614399 128614596 198 browser details YourSeq 40 7 186 2000 84.5% chr7 - 78815786 78815965 180 browser details YourSeq 34 1 53 2000 83.1% chr4 - 133559245 133559460 216 browser details YourSeq 32 5 44 2000 90.0% chr10 - 81539720 81539759 40 browser details YourSeq 31 890 926 2000 91.9% chr18 - 61804945 61804981 37 browser details YourSeq 31 4 44 2000 87.9% chr9 + 114943683 114943723 41 browser details YourSeq 31 1 41 2000 87.9% chr2 + 32104812 32104852 41 browser details YourSeq 30 1 44 2000 84.1% chr15 - 101155345 101155388 44 browser details YourSeq 30 1 44 2000 88.3% chr4 + 117269559 117269601 43 browser details YourSeq 29 8 41 2000 84.4% chr8 - 107283571 107283602 32 browser details YourSeq 29 2 44 2000 83.8% chr15 - 77362020 77362062 43 browser details YourSeq 29 1 42 2000 85.8% chr10 - 127906116 127906439 324 browser details YourSeq 29 2 44 2000 83.8% chr6 + 30002426 30002468 43 browser details YourSeq 29 4 44 2000 93.8% chr5 + 137204661 137204701 41 browser details YourSeq 29 8 44 2000 96.8% chr19 + 59115473 59115509 37 browser details YourSeq 29 1 41 2000 85.4% chr10 + 78159221 78159261 41 browser details YourSeq 29 1 39 2000 87.2% chr10 + 40375775 40375813 39 browser details YourSeq 28 1 44 2000 81.9% chr8 - 3491111 3491154 44 browser details YourSeq 28 6 41 2000 88.9% chr11 - 104300706 104300741 36 Note: The 2000 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 470 1 470 470 100.0% chr7 - 110852359 110852828 470 browser details YourSeq 27 184 218 470 80.7% chr2 - 142962196 142962227 32 browser details YourSeq 26 177 203 470 100.0% chr14 - 34283241 34283722 482 browser details YourSeq 20 107 126 470 100.0% chr7 - 127632216 127632235 20 browser details YourSeq 20 181 200 470 100.0% chr4 - 24574334 24574353 20 Note: The 470 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Lyve1 lymphatic vessel endothelial hyaluronan receptor 1 [ Mus musculus (house mouse) ] Gene ID: 114332, updated on 15-Oct-2019 Gene summary Official Symbol Lyve1 provided by MGI Official Full Name lymphatic vessel endothelial hyaluronan receptor 1 provided by MGI Primary source MGI:MGI:2136348 See related Ensembl:ENSMUSG00000030787 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Xlkd1; Lyve-1; Crsbp-1; 1200012G08Rik Expression Broad expression in lung adult (RPKM 22.3), liver E18 (RPKM 15.1) and 15 other tissues See more Orthologs human all Genomic context Location: 7; 7 E3 See Lyve1 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (110850607..110862953, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (117994121..118006467, complement) Chromosome 7 - NC_000073.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Lyve1 ENSMUSG00000030787 Description lymphatic vessel endothelial hyaluronan receptor 1 [Source:MGI Symbol;Acc:MGI:2136348] Gene Synonyms 1200012G08Rik, Lyve-1, Xlkd1, lymphatic vessel endothelial HA receptor-1 Location Chromosome 7: 110,850,607-110,863,239 reverse strand. GRCm38:CM001000.2 About this gene This gene has 2 transcripts (splice variants), 224 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Lyve1-201 ENSMUST00000033050.4 2893 318aa ENSMUSP00000033050.3 Protein coding CCDS21748 Q8BHC0 TSL:1 GENCODE basic APPRIS P1 Lyve1-202 ENSMUST00000209319.1 802 No protein - Retained intron - - TSL:2 32.63 kb Forward strand 110.85Mb 110.86Mb 110.87Mb Contigs AC184052.2 > Genes (Comprehensive set... < Rnf141-209protein coding < Lyve1-201protein coding < Mrvi1-203protein coding < Rnf141-210protein coding < Lyve1-202retained intron < Mrvi1-201protein coding < Rnf141-201protein coding < Mrvi1-202protein coding < Rnf141-208protein coding < Rnf141-207protein coding < Rnf141-211protein coding < Rnf141-212retained intron Regulatory Build 110.85Mb 110.86Mb 110.87Mb Reverse strand 32.63 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000033050 < Lyve1-201protein coding Reverse strand 12.63 kb ENSMUSP00000033... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily C-type lectin fold SMART Link domain Pfam Link domain PROSITE profiles Link domain PROSITE patterns Link domain PANTHER PTHR10225:SF2 PTHR10225 Gene3D C-type lectin-like/link domain superfamily CDD cd03516 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 318 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.