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Mouse Prn Conditional Knockout Project (CRISPR/Cas9)

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Mouse Prn Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Prn Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Prn conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Prn gene (NCBI Reference Sequence: NM_001278258 ; Ensembl: ENSMUSG00000098754 ) is located on Mouse chromosome 2. 4 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000136783). 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 Prn gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-401J24 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 3 starts from about 0.51% 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: 34078 bp, and the size of intron 3 for 3'-loxP site insertion: 6535 bp. The size of effective cKO region: ~547 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 gRNA region 5' gRNA region 3' 1 3 4 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Prn 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(7047bp) | A(26.41% 1861) | C(23.29% 1641) | T(27.88% 1965) | G(22.42% 1580) 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% chr2 + 131943178 131946177 3000 browser details YourSeq 257 715 1148 3000 91.1% chr3 - 152721688 152722107 420 browser details YourSeq 221 713 1149 3000 89.9% chr7 - 34235238 34235714 477 browser details YourSeq 221 715 1148 3000 87.0% chr5 + 106889204 106889600 397 browser details YourSeq 207 756 1170 3000 90.6% chr1 - 58422270 58422757 488 browser details YourSeq 201 756 1170 3000 89.2% chr11 + 107539252 107539852 601 browser details YourSeq 195 758 980 3000 94.7% chr4 - 108674004 109044428 370425 browser details YourSeq 175 737 978 3000 94.0% chr5 - 123124386 123124843 458 browser details YourSeq 173 788 1150 3000 89.6% chr7 - 16217166 16217610 445 browser details YourSeq 173 927 1150 3000 97.3% chr12 - 110773824 110996157 222334 browser details YourSeq 173 711 1143 3000 94.8% chr1 + 118831527 118832180 654 browser details YourSeq 168 743 1124 3000 87.9% chr7 + 3478647 3479129 483 browser details YourSeq 164 756 980 3000 92.8% chr1 + 72151942 72573478 421537 browser details YourSeq 159 733 978 3000 92.1% chr9 - 100486996 100487374 379 browser details YourSeq 156 737 980 3000 89.4% chr10 - 81118536 81118830 295 browser details YourSeq 153 756 1149 3000 89.3% chr1 + 93744891 93745461 571 browser details YourSeq 151 728 971 3000 93.2% chr8 + 46532258 46532815 558 browser details YourSeq 150 1015 1178 3000 95.1% chr13 - 63315111 63315273 163 browser details YourSeq 150 737 974 3000 84.5% chr11 - 60429832 60430032 201 browser details YourSeq 147 716 883 3000 94.0% chr1 - 122630261 122987689 357429 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% chr2 + 131946725 131949724 3000 browser details YourSeq 30 2788 2838 3000 94.2% chr7 - 42530015 42530066 52 browser details YourSeq 22 2651 2678 3000 89.3% chr17 - 59042846 59042873 28 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Prn prion protein readthrough transcript [ Mus musculus (house mouse) ] Gene ID: 111368, updated on 10-Oct-2019 Gene summary Official Symbol Prn provided by MGI Official Full Name prion protein readthrough transcript provided by MGI Primary source MGI:MGI:97767 See related Ensembl:ENSMUSG00000098754 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 Gm28045; Gm44637; Prnp-Prnd Summary This locus represents naturally occurring readthrough transcription between the neighboring Prnp (major prion protein) and Expression Prnd (prion-like protein doppel) genes on chromosome 2. The readthrough transcript encodes a protein that shares sequence identity with the downstream gene product but its N-terminal region is distinct due to the presence of an in-frame, upstream AUG compared to the downstream gene. [provided by RefSeq, May 2013] Broad expression in testis adult (RPKM 22.5), limb E14.5 (RPKM 10.9) and 17 other tissues See more Genomic context Location: 2 F2; 2 See Prn in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (131909928..131956131) Chromosome 2 - NC_000068.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Prn ENSMUSG00000098754 Description prion protein readthrough transcript [Source:MGI Symbol;Acc:MGI:97767] Gene Synonyms Gm28045, Gm44637 Location Chromosome 2: 131,909,957-131,956,130 forward strand. GRCm38:CM000995.2 About this gene This gene has 2 transcripts (splice variants), 70 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Prn-202 ENSMUST00000136783.1 3370 197aa ENSMUSP00000122345.2 Protein coding CCDS71149 A0A075B668 TSL:2 GENCODE basic APPRIS P1 Prn-201 ENSMUST00000124100.7 2132 197aa ENSMUSP00000116195.2 Protein coding CCDS71149 A0A075B668 TSL:2 GENCODE basic APPRIS P1 66.17 kb Forward strand 131.90Mb 131.92Mb 131.94Mb 131.96Mb Genes Lamr1-ps1-201 >processed pseudogene Prnd-202 >protein coding (Comprehensive set... Prnp-201 >protein coding Prnd-203 >protein coding Prnp-202 >lncRNA Prnd-204 >protein coding Prn-202 >protein coding Prn-201 >protein coding Prnd-201 >protein coding Contigs AL833794.9 > Regulatory Build 131.90Mb 131.92Mb 131.94Mb 131.96Mb Reverse strand 66.17 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000136783 46.17 kb Forward strand Prn-202 >protein coding ENSMUSP00000122... Transmembrane heli... Superfamily Prion/Doppel beta-ribbon domain superfamily Pfam Prion-like protein Doppel Prion/Doppel protein, beta-ribbon domain PANTHER PTHR15506 PTHR15506:SF0 Gene3D Prion/Doppel beta-ribbon domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 197 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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