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Mouse Bet1 Knockout Project (CRISPR/Cas9)

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Mouse Bet1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Bet1 Knockout Project (CRISPR/Cas9) Objective: To create a Bet1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Bet1 gene (NCBI Reference Sequence: NM_009748 ; Ensembl: ENSMUSG00000032757 ) is located on Mouse chromosome 6. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000049166). Exon 1~4 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: Exon 1 starts from about 0.28% of the coding region. Exon 1~4 covers 100.0% of the coding region. The size of effective KO region: ~8910 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 Legends Exon of mouse Bet1 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 start codon 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 stop codon 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. 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(28.4% 568) | C(23.05% 461) | T(30.5% 610) | G(18.05% 361) Note: The 2000 bp section upstream of start codon 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(28.85% 577) | C(16.7% 334) | T(35.7% 714) | G(18.75% 375) Note: The 2000 bp section downstream of stop codon 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% chr6 - 4086796 4088795 2000 browser details YourSeq 23 372 394 2000 100.0% chr7 - 76397716 76397738 23 browser details YourSeq 22 1085 1106 2000 100.0% chr3 - 100595006 100595027 22 browser details YourSeq 22 1777 1800 2000 87.0% chr1 + 15005751 15005773 23 browser details YourSeq 21 698 718 2000 100.0% chr12 + 95294281 95294301 21 browser details YourSeq 20 1104 1127 2000 91.7% chr9 + 18440925 18440948 24 browser details YourSeq 20 633 652 2000 100.0% chr1 + 70522243 70522262 20 Note: The 2000 bp section upstream of start codon 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% chr6 - 4075884 4077883 2000 browser details YourSeq 336 1045 1402 2000 97.0% chr2 + 94663510 94663867 358 browser details YourSeq 336 1045 1402 2000 96.4% chr16 + 47742201 47742557 357 browser details YourSeq 336 1045 1402 2000 97.0% chr15 + 29874500 29874857 358 browser details YourSeq 334 1015 1385 2000 97.5% chr6 - 56406511 56407233 723 browser details YourSeq 334 1045 1396 2000 97.5% chr9 + 92952726 92953077 352 browser details YourSeq 333 1045 1397 2000 97.2% chr10 - 4582724 4583076 353 browser details YourSeq 333 1045 1397 2000 97.2% chr1 - 192999755 193000107 353 browser details YourSeq 333 1045 1397 2000 97.2% chr7 + 51919796 51920148 353 browser details YourSeq 331 1045 1397 2000 96.9% chr2 + 150150704 150151056 353 browser details YourSeq 330 1045 1402 2000 96.1% chr16 - 70742489 70742846 358 browser details YourSeq 330 1045 1402 2000 96.1% chr10 - 73321278 73321635 358 browser details YourSeq 330 1045 1402 2000 96.1% chr3 + 138314072 138314429 358 browser details YourSeq 329 1045 1385 2000 98.3% chr19 - 24728373 24728713 341 browser details YourSeq 329 1045 1397 2000 96.7% chr14 - 97950425 97950777 353 browser details YourSeq 329 1045 1385 2000 98.3% chr1 - 87905131 87905471 341 browser details YourSeq 328 1045 1402 2000 95.9% chr2 - 80493411 80493768 358 browser details YourSeq 328 1045 1402 2000 95.9% chr1 - 98364297 98364654 358 browser details YourSeq 328 1045 1400 2000 96.1% chr5 + 19303346 19303701 356 browser details YourSeq 328 1045 1402 2000 95.9% chr11 + 58511681 58512038 358 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Bet1 Bet1 golgi vesicular membrane trafficking protein [ Mus musculus (house mouse) ] Gene ID: 12068, updated on 12-Aug-2019 Gene summary Official Symbol Bet1 provided by MGI Official Full Name Bet1 golgi vesicular membrane trafficking protein provided by MGI Primary source MGI:MGI:1343104 See related Ensembl:ENSMUSG00000032757 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 Bet-1; AW555236 Expression Ubiquitous expression in limb E14.5 (RPKM 9.5), liver E18 (RPKM 9.3) and 28 other tissues See more Orthologs human all Genomic context Location: 6; 6 A1 See Bet1 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (4076899..4086998, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (4026904..4036927, complement) Chromosome 6 - NC_000072.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Bet1 ENSMUSG00000032757 Description Bet1 golgi vesicular membrane trafficking protein [Source:MGI Symbol;Acc:MGI:1343104] Location Chromosome 6: 4,076,899-4,086,972 reverse strand. GRCm38:CM000999.2 About this gene This gene has 6 transcripts (splice variants), 125 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Bet1-201 ENSMUST00000049166.4 1516 118aa ENSMUSP00000044877.4 Protein coding CCDS19895 O35623 TSL:1 GENCODE basic APPRIS P1 Bet1-203 ENSMUST00000150319.1 2461 No protein - Retained intron - - TSL:1 Bet1-205 ENSMUST00000204806.1 295 No protein - Retained intron - - TSL:2 Bet1-204 ENSMUST00000203612.1 518 No protein - lncRNA - - TSL:3 Bet1-202 ENSMUST00000145696.3 420 No protein - lncRNA - - TSL:2 Bet1-206 ENSMUST00000205191.1 394 No protein - lncRNA - - TSL:3 30.07 kb Forward strand 4.07Mb 4.08Mb 4.09Mb Contigs AC022235.5 > Genes (Comprehensive set... < Bet1-201protein coding < Bet1-204lncRNA < Bet1-205retained intron < Bet1-203retained intron < Bet1-202lncRNA < Bet1-206lncRNA Regulatory Build 4.07Mb 4.08Mb 4.09Mb Reverse strand 30.07 kb Regulation Legend 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: ENSMUST00000049166 < Bet1-201protein coding Reverse strand 10.07 kb ENSMUSP00000044... Transmembrane heli... Low complexity (Seg) Superfamily SSF58038 SMART Target SNARE coiled-coil homology domain PROSITE profiles Target SNARE coiled-coil homology domain PANTHER BET1-like protein PTHR12791:SF53 Gene3D 1.20.5.110 CDD BET1, SNARE domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 10 20 30 40 50 60 70 80 90 100 118 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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