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Mouse Mon2 Knockout Project (CRISPR/Cas9)

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Mouse Mon2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mon2 Knockout Project (CRISPR/Cas9) Objective: To create a Mon2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mon2 gene (NCBI Reference Sequence: NM_001163024 ; Ensembl: ENSMUSG00000034602 ) is located on Mouse chromosome 10. 36 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 36 (Transcript: ENSMUST00000073792). Exon 3~6 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 3 starts from about 3.42% of the coding region. Exon 3~6 covers 9.43% of the coding region. The size of effective KO region: ~9338 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 6 36 Legends Exon of mouse Mon2 Knockout region Page 2 of 9 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 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 618 bp section downstream of Exon 6 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 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(28.3% 566) | C(18.15% 363) | T(34.4% 688) | G(19.15% 383) Note: The 2000 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(618bp) | A(31.55% 195) | C(16.5% 102) | T(32.69% 202) | G(19.26% 119) Note: The 618 bp section downstream of Exon 6 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 9 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% chr10 - 123052313 123054312 2000 browser details YourSeq 33 778 829 2000 74.4% chr6 - 22771593 22771636 44 browser details YourSeq 32 898 947 2000 82.0% chr13 - 79367862 79367911 50 browser details YourSeq 29 774 807 2000 94.0% chr10 - 107651411 107651446 36 browser details YourSeq 28 776 807 2000 93.8% chr19 - 32537814 32537845 32 browser details YourSeq 28 787 830 2000 73.4% chr12 + 110216365 110216400 36 browser details YourSeq 27 770 801 2000 93.4% chr2 - 101661686 101661717 32 browser details YourSeq 27 897 933 2000 86.5% chr15 + 89799485 89799521 37 browser details YourSeq 26 917 942 2000 100.0% chr2 - 105989691 105989716 26 browser details YourSeq 24 922 951 2000 90.0% chr3 - 17043507 17043536 30 browser details YourSeq 23 779 807 2000 89.7% chr10 + 108092559 108092587 29 browser details YourSeq 23 917 939 2000 100.0% chr1 + 112831556 112831578 23 browser details YourSeq 22 787 808 2000 100.0% chr2 + 161512871 161512892 22 browser details YourSeq 22 916 937 2000 100.0% chr12 + 52581976 52581997 22 browser details YourSeq 21 1245 1271 2000 88.9% chr4 - 50289075 50289101 27 browser details YourSeq 21 1336 1356 2000 100.0% chr18 + 49890531 49890551 21 Note: The 2000 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 618 1 618 618 100.0% chr10 - 123042357 123042974 618 browser details YourSeq 30 367 415 618 71.9% chr14 - 46075930 46075964 35 browser details YourSeq 28 512 551 618 91.0% chr15 - 56951869 56951913 45 browser details YourSeq 26 44 74 618 93.4% chr1 + 30684916 30684947 32 browser details YourSeq 23 312 340 618 80.0% chr14 - 58671805 58671830 26 browser details YourSeq 23 530 553 618 100.0% chr11 - 92826091 92826127 37 browser details YourSeq 21 136 160 618 92.0% chr13 + 22679081 22679105 25 browser details YourSeq 20 108 129 618 95.5% chr14 - 101521105 101521126 22 browser details YourSeq 20 536 555 618 100.0% chr10 - 102421915 102421934 20 browser details YourSeq 20 128 149 618 95.5% chr13 + 29478346 29478367 22 browser details YourSeq 20 76 95 618 100.0% chr11 + 40454739 40454758 20 Note: The 618 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Mon2 MON2 homolog, regulator of endosome to Golgi trafficking [ Mus musculus (house mouse) ] Gene ID: 67074, updated on 12-Aug-2019 Gene summary Official Symbol Mon2 provided by MGI Official Full Name MON2 homolog, regulator of endosome to Golgi trafficking provided by MGI Primary source MGI:MGI:1914324 See related Ensembl:ENSMUSG00000034602 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 Sf21; AW495628; mKIAA1040; 2610528O22Rik Expression Ubiquitous expression in limb E14.5 (RPKM 7.0), genital fat pad adult (RPKM 6.4) and 28 other tissues See more Orthologs human all Genomic context Location: 10; 10 D2 See Mon2 in Genome Data Viewer Exon count: 38 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (122992060..123076505, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (122429117..122513561, complement) Chromosome 10 - NC_000076.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 12 transcripts Gene: Mon2 ENSMUSG00000034602 Description MON2 homolog, regulator of endosome to Golgi trafficking [Source:MGI Symbol;Acc:MGI:1914324] Gene Synonyms 2610528O22Rik, SF21 Location Chromosome 10: 122,992,060-123,076,505 reverse strand. GRCm38:CM001003.2 About this gene This gene has 12 transcripts (splice variants), 199 orthologues, 15 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mon2-202 ENSMUST00000073792.10 9301 1715aa ENSMUSP00000073462.3 Protein coding CCDS48707 Q80TL7 TSL:5 GENCODE basic APPRIS ALT2 Mon2-201 ENSMUST00000037557.8 5511 1708aa ENSMUSP00000037568.7 Protein coding CCDS24216 Q80TL7 TSL:1 GENCODE basic APPRIS P3 Mon2-203 ENSMUST00000170935.8 5302 1709aa ENSMUSP00000131052.1 Protein coding CCDS48708 B9EKJ3 TSL:1 GENCODE basic APPRIS ALT2 Mon2-207 ENSMUST00000219203.1 1844 436aa ENSMUSP00000151951.1 Protein coding - A0A1W2P878 TSL:1 GENCODE basic Mon2-206 ENSMUST00000219001.1 4053 No protein - Retained intron - - TSL:1 Mon2-212 ENSMUST00000222536.1 3978 No protein - Retained intron - - TSL:5 Mon2-208 ENSMUST00000219241.1 882 No protein - Retained intron - - TSL:2 Mon2-211 ENSMUST00000220201.1 882 No protein - Retained intron - - TSL:3 Mon2-205 ENSMUST00000218735.1 765 No protein - Retained intron - - TSL:2 Mon2-210 ENSMUST00000219515.1 481 No protein - Retained intron - - TSL:3 Mon2-209 ENSMUST00000219290.1 459 No protein - Retained intron - - TSL:2 Mon2-204 ENSMUST00000218253.1 225 No protein - lncRNA - - TSL:3 Page 7 of 9 https://www.alphaknockout.com 104.45 kb Forward strand 123.00Mb 123.02Mb 123.04Mb 123.06Mb 123.08Mb Contigs < AC153911.5 AC153021.2 > Genes (Comprehensive set... < D630033A02Rik-202lncRNA < Mon2-205retained intron< Mon2-206retained intron < D630033A02Rik-201transcribed processed pseudogene< Mon2-210retained intron < Mon2-207protein coding < Mirlet7i-201miRNA < Mon2-208retained intron < Mon2-204lncRNA < Mon2-202protein coding < Mon2-212retained intron < Mon2-201protein coding < Mon2-209retained intron < Mon2-211retained intron < Mon2-203protein coding Regulatory Build 123.00Mb 123.02Mb 123.04Mb 123.06Mb 123.08Mb Reverse strand 104.45 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000073792 < Mon2-202protein coding Reverse strand 84.45 kb ENSMUSP00000073... MobiDB lite Low complexity (Seg) Superfamily Armadillo-type fold Pfam Mon2, dimerisation and cyclophilin-binding domain Sec7, C-terminal Guanine nucleotide exchange factor, N-terminal Mon2, C-terminal PANTHER PTHR10663 Protein Mon2-like All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1400 1715 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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