https://www.alphaknockout.com Mouse Zfyve1 Knockout Project (CRISPR/Cas9) Objective: To create a Zfyve1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Zfyve1 gene (NCBI Reference Sequence: NM_183154 ; Ensembl: ENSMUSG00000042628 ) is located on Mouse chromosome 12. 12 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000048319). Exon 2 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: Exon 2 starts from the coding region. Exon 2 covers 20.72% of the coding region. The size of effective KO region: ~925 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 12 Legends Exon of mouse Zfyve1 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 1674 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(1674bp) | A(23.36% 391) | C(19.83% 332) | T(33.15% 555) | G(23.66% 396) Note: The 1674 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(2000bp) | A(24.65% 493) | C(20.45% 409) | T(31.85% 637) | G(23.05% 461) Note: The 2000 bp section downstream 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. 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 1674 1 1674 1674 100.0% chr12 - 83594881 83596554 1674 browser details YourSeq 138 739 977 1674 94.9% chr9 - 72126692 72126953 262 browser details YourSeq 130 768 977 1674 96.5% chr9 - 22209057 22359049 149993 browser details YourSeq 113 743 958 1674 92.6% chr4 + 137834166 137969782 135617 browser details YourSeq 103 764 977 1674 93.4% chr10 - 128825284 128825684 401 browser details YourSeq 103 753 977 1674 95.6% chr10 + 88709577 88709910 334 browser details YourSeq 100 869 977 1674 96.4% chr10 + 128747235 128747355 121 browser details YourSeq 99 742 971 1674 86.5% chr8 - 111938252 111938461 210 browser details YourSeq 97 866 977 1674 90.0% chr11 - 48652312 48652411 100 browser details YourSeq 96 870 977 1674 91.3% chr6 - 24660277 24660379 103 browser details YourSeq 96 872 977 1674 92.1% chr4 - 57103772 57103872 101 browser details YourSeq 96 867 978 1674 96.2% chr1 - 81849146 81849266 121 browser details YourSeq 96 756 977 1674 90.6% chr9 + 44382314 44382842 529 browser details YourSeq 95 867 977 1674 91.0% chr18 + 36393766 36393865 100 browser details YourSeq 95 870 977 1674 96.2% chr13 + 3873650 3874044 395 browser details YourSeq 93 867 977 1674 88.3% chr8 + 22412884 22412985 102 browser details YourSeq 93 872 977 1674 96.1% chr4 + 109069602 109069715 114 browser details YourSeq 92 873 977 1674 96.0% chr7 + 127649526 127649634 109 browser details YourSeq 92 867 977 1674 95.1% chr10 + 89852343 89852645 303 browser details YourSeq 91 879 977 1674 97.0% chr9 - 90116262 90116533 272 Note: The 1674 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 2000 1 2000 2000 100.0% chr12 - 83592398 83594397 2000 browser details YourSeq 190 1351 1919 2000 83.7% chr6 - 122418622 122418896 275 browser details YourSeq 188 1348 1919 2000 90.2% chr1 + 16658414 17070316 411903 browser details YourSeq 185 1325 1919 2000 84.9% chr12 - 81542120 81542396 277 browser details YourSeq 183 1344 1919 2000 89.3% chr4 - 149755169 149755731 563 browser details YourSeq 182 1325 1692 2000 94.7% chr3 + 107339553 107340079 527 browser details YourSeq 178 1347 1567 2000 92.8% chr2 - 39024724 39114442 89719 browser details YourSeq 175 1325 1562 2000 94.0% chr17 - 83560368 83560606 239 browser details YourSeq 174 1325 1564 2000 88.5% chr17 - 26191699 26191926 228 browser details YourSeq 170 1352 1567 2000 93.0% chr7 - 19480481 19481011 531 browser details YourSeq 170 1348 1601 2000 90.9% chr4 + 138009236 138009566 331 browser details YourSeq 169 1348 1561 2000 94.8% chr15 + 83147614 83148034 421 browser details YourSeq 163 1351 1564 2000 91.8% chr19 - 37463031 37463251 221 browser details YourSeq 161 994 1485 2000 87.1% chr13 - 40910018 40910365 348 browser details YourSeq 154 1325 1618 2000 87.0% chr11 + 58169045 58169236 192 browser details YourSeq 153 1329 1616 2000 95.8% chr11 + 83376625 83377224 600 browser details YourSeq 151 1352 1579 2000 85.5% chr9 - 44394171 44394393 223 browser details YourSeq 145 1267 1482 2000 91.0% chr11 - 114674771 114674965 195 browser details YourSeq 144 1325 1485 2000 94.0% chr17 - 6226699 6226852 154 browser details YourSeq 144 1325 1617 2000 86.3% chr5 + 88731715 88731890 176 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Zfyve1 zinc finger, FYVE domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 217695, updated on 24-Oct-2019 Gene summary Official Symbol Zfyve1 provided by MGI Official Full Name zinc finger, FYVE domain containing 1 provided by MGI Primary source MGI:MGI:3026685 See related Ensembl:ENSMUSG00000042628 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 DFCP1; TAFF1; ZNFN2A1; mKIAA1589; G630053K23 Expression Ubiquitous expression in testis adult (RPKM 23.4), liver adult (RPKM 10.7) and 28 other tissues See more Orthologs human all Genomic context Location: 12; 12 D1 See Zfyve1 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (83546556..83597245, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (84887891..84938097, complement) Chromosome 12 - NC_000078.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Zfyve1 ENSMUSG00000042628 Description zinc finger, FYVE domain containing 1 [Source:MGI Symbol;Acc:MGI:3026685] Location Chromosome 12: 83,546,558-83,597,222 reverse strand. GRCm38:CM001005.2 About this gene This gene has 6 transcripts (splice variants), 255 orthologues, 12 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Zfyve1-203 ENSMUST00000221919.1 4420 777aa ENSMUSP00000152501.1 Protein coding CCDS26028 Q810J8 TSL:1 GENCODE basic APPRIS P1 Zfyve1-201 ENSMUST00000048319.5 4036 777aa ENSMUSP00000042224.4 Protein coding CCDS26028 Q810J8 TSL:1 GENCODE basic APPRIS P1 Zfyve1-204 ENSMUST00000222448.1 678 175aa ENSMUSP00000152864.1 Protein coding - A0A1Y7VKG0 CDS 3' incomplete TSL:3 Zfyve1-205 ENSMUST00000223222.1 4352 No protein - Retained intron - - TSL:NA Zfyve1-206 ENSMUST00000223380.1 465 No protein - Retained intron - - TSL:2 Zfyve1-202 ENSMUST00000221228.1 375 No protein - Retained intron - - TSL:2 70.67 kb Forward strand 83.54Mb 83.56Mb 83.58Mb 83.60Mb Genes Dcaf4-201 >protein coding (Comprehensive set... Dcaf4-208 >protein coding Dcaf4-203 >retained intron Contigs AC139382.3 > < AC055772.16 Genes (Comprehensive set... < Zfyve1-203protein coding < Zfyve1-201protein coding < Zfyve1-202retained intron < Zfyve1-205retained intron < Zfyve1-204protein coding < Zfyve1-206retained intron Regulatory Build 83.54Mb 83.56Mb 83.58Mb 83.60Mb Reverse strand 70.67 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000048319 < Zfyve1-201protein coding Reverse strand 50.37 kb ENSMUSP00000042... Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase Zinc finger, FYVE/PHD-type SMART FYVE zinc finger Pfam FYVE zinc finger PROSITE profiles Zinc finger, FYVE-related PANTHER Zinc finger FYVE domain-containing protein 1 PTHR46624:SF3 Gene3D 3.40.50.300 Zinc finger, RING/FYVE/PHD-type CDD cd01851 cd15734 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 80 160 240 320 400 480 560 640 777 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.