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Mouse Nav2 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Nav2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Nav2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Nav2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nav2 gene (NCBI Reference Sequence: NM_175272 ; Ensembl: ENSMUSG00000052512 ) is located on Mouse chromosome 7. 39 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 39 (Transcript: ENSMUST00000184945). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nav2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-254C4 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: Homozygous null mice display impaired olfaction and hearing, increased latency in a hot plate test, degeneration of the optic nerve, decreased exploration in new environments, and weight loss. Exon 3 starts from about 5.17% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 10101 bp, and the size of intron 3 for 3'-loxP site insertion: 25713 bp. The size of effective cKO region: ~553 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 3 39 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Nav2 Homology arm cKO region loxP site Page 2 of 8 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7053bp) | A(24.85% 1753) | C(23.08% 1628) | T(28.7% 2024) | G(23.37% 1648) 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 8 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% chr7 + 49369068 49372067 3000 browser details YourSeq 49 1539 1600 3000 93.0% chr14 + 122458984 122459047 64 browser details YourSeq 47 1539 1600 3000 91.3% chr11 - 56460355 56460418 64 browser details YourSeq 46 1520 1586 3000 92.6% chr1 + 136737288 136737389 102 browser details YourSeq 43 1546 1647 3000 90.6% chr3 + 95082980 95083131 152 browser details YourSeq 41 1540 1586 3000 93.7% chr15 + 102800641 102800687 47 browser details YourSeq 39 1544 1586 3000 90.5% chr2 + 32558516 32558557 42 browser details YourSeq 38 1530 1578 3000 93.2% chr1 - 59433465 59433523 59 browser details YourSeq 37 1544 1604 3000 95.2% chr7 - 78866927 78866991 65 browser details YourSeq 37 1524 1582 3000 93.1% chrX + 37181132 37181536 405 browser details YourSeq 36 1545 1586 3000 92.9% chr7 - 79233508 79233549 42 browser details YourSeq 36 1538 1577 3000 92.2% chr16 - 20113121 20113159 39 browser details YourSeq 36 1545 1586 3000 92.9% chr9 + 71528461 71528502 42 browser details YourSeq 35 1544 1584 3000 92.7% chr10 + 57646423 57646463 41 browser details YourSeq 34 1547 1586 3000 92.5% chr17 - 45354808 45354847 40 browser details YourSeq 34 1547 1586 3000 92.5% chr11 - 113425218 113425257 40 browser details YourSeq 34 1547 1586 3000 92.5% chr1 - 110921097 110921136 40 browser details YourSeq 34 1547 1586 3000 92.5% chr1 - 54528788 54528827 40 browser details YourSeq 34 1547 1586 3000 92.5% chrX + 22771160 22771199 40 browser details YourSeq 34 1544 1586 3000 85.0% chr9 + 80163680 80163720 41 Note: The 3000 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 3000 1 3000 3000 100.0% chr7 + 49372621 49375620 3000 browser details YourSeq 40 620 671 3000 97.7% chr3 + 18765623 18765816 194 browser details YourSeq 37 620 670 3000 85.8% chr5 - 128910918 128910966 49 browser details YourSeq 37 620 671 3000 97.5% chr3 - 156642434 156642486 53 browser details YourSeq 33 620 669 3000 97.3% chr5 - 111730242 111730398 157 browser details YourSeq 33 620 671 3000 97.2% chr5 + 119728815 119728950 136 browser details YourSeq 32 620 667 3000 97.1% chr2 - 171534765 171534812 48 browser details YourSeq 31 620 669 3000 97.0% chr13 - 40262702 40262765 64 browser details YourSeq 31 620 669 3000 86.2% chr17 + 81860762 81860809 48 browser details YourSeq 31 627 665 3000 89.8% chr17 + 63899798 63899836 39 browser details YourSeq 30 627 669 3000 75.0% chr3 + 68704097 68704129 33 browser details YourSeq 29 620 666 3000 68.8% chr17 + 13388494 13388526 33 browser details YourSeq 28 636 667 3000 96.7% chr3 - 79245573 79245606 34 browser details YourSeq 24 643 669 3000 84.0% chr17 - 43491204 43491228 25 browser details YourSeq 22 1586 1627 3000 76.2% chr10 - 46897280 46897321 42 browser details YourSeq 21 620 644 3000 92.0% chr5 - 146725903 146725927 25 browser details YourSeq 20 620 639 3000 100.0% chr15 + 94976470 94976489 20 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Nav2 neuron navigator 2 [ Mus musculus (house mouse) ] Gene ID: 78286, updated on 12-Aug-2019 Gene summary Official Symbol Nav2 provided by MGI Official Full Name neuron navigator 2 provided by MGI Primary source MGI:MGI:2183691 See related Ensembl:ENSMUSG00000052512 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 HELAD1; RAINB1; RAINB2; POMFIL2; Unc53H2; AU067780; BQ960892; mKIAA3015; 5330421F07Rik Expression Ubiquitous expression in genital fat pad adult (RPKM 5.2), lung adult (RPKM 4.6) and 28 other tissues See more Orthologs human all Genomic context Location: 7; 7 B4 See Nav2 in Genome Data Viewer Exon count: 53 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (48959063..49610090) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (56214443..56865458) Chromosome 7 - NC_000073.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 18 transcripts Gene: Nav2 ENSMUSG00000052512 Description neuron navigator 2 [Source:MGI Symbol;Acc:MGI:2183691] Gene Synonyms 5330421F07Rik, HELAD1, POMFIL2, RAINB2, Rainb1, Unc53H2 Location Chromosome 7: 48,908,716-49,610,090 forward strand. GRCm38:CM001000.2 About this gene This gene has 18 transcripts (splice variants), 216 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 25 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nav2- ENSMUST00000184945.7 10665 2432aa ENSMUSP00000139045.1 Protein coding CCDS71973 V9GX91 TSL:5 216 GENCODE basic APPRIS P4 Nav2- ENSMUST00000183659.7 7765 2371aa ENSMUSP00000139309.1 Protein coding CCDS71972 V9GXT3 TSL:5 204 GENCODE basic APPRIS ALT2 Nav2- ENSMUST00000064395.12 11317 2432aa ENSMUSP00000067448.6 Protein coding - E9Q842 TSL:5 202 GENCODE basic APPRIS ALT2 Nav2- ENSMUST00000184109.1 890 296aa ENSMUSP00000138846.1 Protein coding - V9GWV3 CDS 5' and 3' 207 incomplete TSL:3 Nav2- ENSMUST00000207743.1 431 116aa ENSMUSP00000159080.1 Protein coding - - CDS 3' incomplete 218 TSL:3 Nav2- ENSMUST00000184124.7 5339 103aa ENSMUSP00000139312.1 Nonsense mediated - V9GXT5 TSL:1 208 decay Nav2- ENSMUST00000183531.1 4144 No - Retained intron - - TSL:1 203 protein Nav2- ENSMUST00000184928.7 3439 No - Retained intron - - TSL:1 215 protein Nav2- ENSMUST00000064383.6 3224 No - Retained intron - - TSL:1 201 protein Nav2- ENSMUST00000184544.1 2839 No - Retained intron - - TSL:NA 213 protein Nav2- ENSMUST00000184809.7 2265 No - Retained intron - - TSL:1 214 protein Nav2- ENSMUST00000184462.1 1664 No - Retained intron - - TSL:NA 211 protein Nav2- ENSMUST00000184471.1 1584 No - Retained intron - - TSL:NA 212 protein Nav2- ENSMUST00000184102.1 1466 No - Retained intron - - TSL:NA 206 protein Nav2- ENSMUST00000183715.1 1290 No - Retained intron - - TSL:2 205 protein Nav2- ENSMUST00000184955.7 1920 No - lncRNA - - TSL:1 217 protein Nav2- ENSMUST00000184155.1 1495 No - lncRNA - - TSL:1 209 protein Nav2- ENSMUST00000184336.1 483 No - lncRNA - - TSL:3 210 protein Page 6 of 8 https://www.alphaknockout.com 721.38 kb Forward strand 49.0Mb 49.2Mb 49.4Mb 49.6Mb Genes (Comprehensive set... Gm2788-205 >lncRNA Gm32849-201 >lncRNA Gm37613-201 >TEC Nav2-215 >retained intron Nav2-209 >lncRNA Nav2-213 >retained intron Gm2788-206 >lncRNA Nav2-202 >protein coding Gm2788-207 >lncRNA Nav2-214
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