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Mouse Vps36 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Vps36 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Vps36 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Vps36 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Vps36 gene (NCBI Reference Sequence: NM_027338 ; Ensembl: ENSMUSG00000031479 ) is located on Mouse chromosome 8. 14 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 14 (Transcript: ENSMUST00000033866). Exon 4~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Vps36 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-299I15 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 4 starts from about 20.47% of the coding region. The knockout of Exon 4~6 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 3322 bp, and the size of intron 6 for 3'-loxP site insertion: 2325 bp. The size of effective cKO region: ~2777 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 4 5 6 14 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Vps36 Homology arm cKO region loxP site Page 2 of 7 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(9277bp) | A(26.72% 2479) | C(19.12% 1774) | T(32.11% 2979) | G(22.04% 2045) 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 7 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% chr8 + 22202418 22205417 3000 browser details YourSeq 129 592 755 3000 97.1% chrX - 118456406 118456654 249 browser details YourSeq 122 602 755 3000 98.5% chr3 - 90769481 90769723 243 browser details YourSeq 117 853 1208 3000 87.8% chr6 + 93762828 93763203 376 browser details YourSeq 117 1085 1922 3000 77.3% chr2 + 144870479 144871191 713 browser details YourSeq 116 592 731 3000 90.0% chr9 + 84216947 84217066 120 browser details YourSeq 116 597 732 3000 99.2% chr10 + 105970790 105970954 165 browser details YourSeq 115 592 732 3000 88.7% chr6 + 13229540 13229662 123 browser details YourSeq 115 601 732 3000 94.1% chr3 + 121016844 121016969 126 browser details YourSeq 115 599 732 3000 98.4% chr13 + 62341509 62341706 198 browser details YourSeq 114 627 755 3000 99.2% chr15 + 22676864 22677091 228 browser details YourSeq 113 596 732 3000 90.6% chr8 - 33014278 33014394 117 browser details YourSeq 113 597 731 3000 91.3% chr2 + 149589380 149589493 114 browser details YourSeq 113 602 732 3000 91.4% chr1 + 193245492 193245607 116 browser details YourSeq 112 627 755 3000 98.3% chr9 - 18337306 18337533 228 browser details YourSeq 112 629 755 3000 99.2% chr18 + 88128014 88128239 226 browser details YourSeq 111 628 755 3000 98.3% chr12 - 11235332 11235557 226 browser details YourSeq 111 621 732 3000 100.0% chr17 + 52077231 52077344 114 browser details YourSeq 111 596 732 3000 89.5% chr13 + 50149831 50149944 114 browser details YourSeq 111 622 732 3000 100.0% chr10 + 8144806 8144916 111 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% chr8 + 22208195 22211194 3000 browser details YourSeq 178 2806 3000 3000 95.9% chr4 - 11210071 11210388 318 browser details YourSeq 175 2804 2992 3000 95.3% chrX + 105284474 105284661 188 browser details YourSeq 175 2802 2991 3000 96.4% chr14 + 52259420 52259613 194 browser details YourSeq 173 2806 3000 3000 95.4% chr18 - 8098776 8098991 216 browser details YourSeq 173 2806 2992 3000 94.6% chr10 + 81717177 81717361 185 browser details YourSeq 173 2806 2992 3000 94.6% chr10 + 81876287 81876471 185 browser details YourSeq 172 2802 2991 3000 95.8% chr5 + 124500287 124500503 217 browser details YourSeq 172 2801 2993 3000 94.9% chr12 + 28674212 28674405 194 browser details YourSeq 170 2802 2992 3000 95.3% chr6 - 120924857 120925054 198 browser details YourSeq 170 2806 2991 3000 95.7% chr19 - 29681055 29681240 186 browser details YourSeq 170 2806 2991 3000 95.7% chr3 + 138504544 138504729 186 browser details YourSeq 169 2803 2994 3000 94.3% chr2 - 9900241 9900435 195 browser details YourSeq 169 2797 2995 3000 93.0% chr16 - 35856205 35856428 224 browser details YourSeq 168 2801 2989 3000 94.7% chr2 - 69638246 69638434 189 browser details YourSeq 168 2786 2992 3000 89.3% chr13 - 109207252 109207448 197 browser details YourSeq 168 2804 2992 3000 95.7% chr13 - 14035425 14035618 194 browser details YourSeq 167 2806 2992 3000 93.6% chrX - 12881435 12881620 186 browser details YourSeq 167 2806 2992 3000 94.7% chr7 - 80567627 80567813 187 browser details YourSeq 167 2809 2993 3000 94.1% chr5 - 138109917 138110100 184 Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Vps36 vacuolar protein sorting 36 [ Mus musculus (house mouse) ] Gene ID: 70160, updated on 12-Aug-2019 Gene summary Official Symbol Vps36 provided by MGI Official Full Name vacuolar protein sorting 36 provided by MGI Primary source MGI:MGI:1917410 See related Ensembl:ENSMUSG00000031479 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 Eap45; 1700010A24Rik; 2210415M20Rik; 2810408E15Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 12.8), bladder adult (RPKM 12.7) and 28 other tissues See more Orthologs human all Genomic context Location: 8; 8 A2 See Vps36 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (22192833..22220953) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (23303332..23329069) Chromosome 8 - NC_000074.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Vps36 ENSMUSG00000031479 Description vacuolar protein sorting 36 [Source:MGI Symbol;Acc:MGI:1917410] Gene Synonyms 1700010A24Rik, 2210415M20Rik, 2810408E15Rik, Eap45 Location Chromosome 8: 22,192,809-22,220,843 forward strand. GRCm38:CM001001.2 About this gene This gene has 2 transcripts (splice variants), 202 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Vps36-201 ENSMUST00000033866.8 3574 386aa ENSMUSP00000033866.8 Protein coding CCDS22171 Q3TSR1 Q91XD6 TSL:1 GENCODE basic APPRIS P1 Vps36-202 ENSMUST00000153949.1 528 No protein - Retained intron - - TSL:1 48.03 kb Forward strand 22.19Mb 22.20Mb 22.21Mb 22.22Mb 22.23Mb Genes (Comprehensive set... Vps36-201 >protein coding Thsd1-201 >protein coding Vps36-202 >retained intron Thsd1-202 >protein coding Thsd1-203 >protein coding Contigs AC117665.6 > Genes < Ckap2-201protein coding (Comprehensive set... Regulatory Build 22.19Mb 22.20Mb 22.21Mb 22.22Mb 22.23Mb Reverse strand 48.03 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 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000033866 28.04 kb Forward strand Vps36-201 >protein coding ENSMUSP00000033... PDB-ENSP mappings Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 Winged helix DNA-binding domain superfamily Pfam Vacuolar protein sorting protein 36, GLUE domain Snf8/Vps36 family PROSITE profiles Vacuolar protein sorting protein 36, GLUE domain PANTHER Vacuolar protein sorting protein 36 Gene3D PH-like domain superfamily Winged helix-like DNA-binding domain superfamily CDD cd13226 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 386 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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