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Mouse Ppp1r14d Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ppp1r14d Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ppp1r14d Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ppp1r14d conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ppp1r14d gene (NCBI Reference Sequence: NM_001290796 ; Ensembl: ENSMUSG00000027317 ) is located on Mouse chromosome 2. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000110820). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp1r14d gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-39N18 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 2 starts from about 49.33% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1720 bp, and the size of intron 2 for 3'-loxP site insertion: 8909 bp. The size of effective cKO region: ~565 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 2 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Ppp1r14d 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(7065bp) | A(24.33% 1719) | C(24.09% 1702) | T(29.6% 2091) | G(21.98% 1553) 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 - 119228072 119231071 3000 browser details YourSeq 116 9 2883 3000 94.0% chr1 + 153489675 153668756 179082 browser details YourSeq 100 2785 2896 3000 92.7% chr11 + 35968853 35968962 110 browser details YourSeq 98 2779 2883 3000 97.2% chr12 - 23718631 23718736 106 browser details YourSeq 92 2789 2896 3000 95.3% chr14 - 103928290 103928466 177 browser details YourSeq 87 2791 2899 3000 91.2% chr1 + 12522168 12522267 100 browser details YourSeq 72 1 274 3000 81.3% chr19 - 6002890 6003120 231 browser details YourSeq 64 2793 2860 3000 97.1% chr10 - 9481683 9481750 68 browser details YourSeq 62 1 67 3000 97.0% chr15 + 38559416 38559484 69 browser details YourSeq 60 1 71 3000 95.5% chr5 + 65541496 65541572 77 browser details YourSeq 60 1 71 3000 95.5% chr17 + 6314520 6314595 76 browser details YourSeq 60 1 71 3000 95.5% chr17 + 6652914 6652989 76 browser details YourSeq 58 1 71 3000 94.0% chr17 - 6432243 6432318 76 browser details YourSeq 56 7 65 3000 98.4% chr3 - 93567283 93567345 63 browser details YourSeq 56 1 67 3000 92.6% chr1 + 151455915 151455986 72 browser details YourSeq 55 1 56 3000 100.0% chr9 - 96769061 96769117 57 browser details YourSeq 55 1 65 3000 93.0% chr7 - 141306690 141306752 63 browser details YourSeq 55 1 55 3000 100.0% chr5 - 76268053 76268107 55 browser details YourSeq 55 1 59 3000 96.7% chr13 - 65097621 65097679 59 browser details YourSeq 54 1 56 3000 98.3% chr9 - 61329036 61329091 56 Note: The 3000 bp section upstream of Exon 2 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 - 119224507 119227506 3000 browser details YourSeq 210 1212 1540 3000 83.7% chr1 + 6725765 6726099 335 browser details YourSeq 209 1222 1535 3000 87.8% chr10 - 73255021 73255344 324 browser details YourSeq 199 1133 1540 3000 86.8% chr11 - 19438061 19879400 441340 browser details YourSeq 194 1133 1540 3000 79.5% chr6 + 141330754 141331096 343 browser details YourSeq 193 1137 1540 3000 79.2% chr9 - 83666837 83667154 318 browser details YourSeq 191 1133 1540 3000 84.8% chr17 + 49821758 49822167 410 browser details YourSeq 183 1135 1540 3000 82.2% chr8 + 84320241 84320627 387 browser details YourSeq 182 1133 1521 3000 78.1% chr6 + 25080754 25081077 324 browser details YourSeq 172 1133 1543 3000 75.5% chr16 + 4249836 4250144 309 browser details YourSeq 172 1133 1541 3000 79.4% chr1 + 164807264 164807612 349 browser details YourSeq 169 984 1540 3000 88.9% chr7 + 101374921 101375506 586 browser details YourSeq 168 1128 1538 3000 79.3% chr12 + 109269131 109269523 393 browser details YourSeq 166 1144 1540 3000 82.4% chr7 - 128934988 128935347 360 browser details YourSeq 166 1171 1541 3000 81.8% chr12 + 102911353 102911709 357 browser details YourSeq 165 1144 1532 3000 78.7% chrX - 7338240 7338573 334 browser details YourSeq 165 1173 1540 3000 79.7% chr1_GL456221_random - 111376 111690 315 browser details YourSeq 165 1173 1540 3000 80.0% chr1 - 85482466 85482780 315 browser details YourSeq 164 1133 1540 3000 79.8% chr2 - 23021396 23021721 326 browser details YourSeq 160 964 1521 3000 83.2% chr10 + 10645826 10646253 428 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ppp1r14d protein phosphatase 1, regulatory inhibitor subunit 14D [ Mus musculus (house mouse) ] Gene ID: 72112, updated on 12-Aug-2019 Gene summary Official Symbol Ppp1r14d provided by MGI Official Full Name protein phosphatase 1, regulatory inhibitor subunit 14D provided by MGI Primary source MGI:MGI:1919362 See related Ensembl:ENSMUSG00000027317 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 GBPI-1; Ppp1r14; 2010107K19Rik Expression Biased expression in large intestine adult (RPKM 112.3), small intestine adult (RPKM 50.9) and 2 other tissues See more Orthologs human all Genomic context Location: 2; 2 E5 See Ppp1r14d in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (119218119..119230076, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (119043859..119055601, complement) Chromosome 2 - NC_000068.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Ppp1r14d ENSMUSG00000027317 Description protein phosphatase 1, regulatory inhibitor subunit 14D [Source:MGI Symbol;Acc:MGI:1919362] Gene Synonyms 2010107K19Rik Location Chromosome 2: 119,218,119-119,229,906 reverse strand. GRCm38:CM000995.2 About this gene This gene has 2 transcripts (splice variants), 179 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ppp1r14d-202 ENSMUST00000110820.2 790 175aa ENSMUSP00000106444.2 Protein coding CCDS71118 Q7TT52 TSL:1 GENCODE basic Ppp1r14d-201 ENSMUST00000076084.5 787 146aa ENSMUSP00000075453.5 Protein coding CCDS16596 Q7TT52 TSL:1 GENCODE basic APPRIS P1 31.79 kb Forward strand 119.21Mb 119.22Mb 119.23Mb Genes Zfyve19-208 >lncRNA Zfyve19-203 >retained intron Spint1-201 >protein coding (Comprehensive set... Zfyve19-201 >protein coding Spint1-202 >protein coding Zfyve19-206 >lncRNA Zfyve19-207 >retained intron Spint1-203 >protein coding Zfyve19-202 >protein coding Zfyve19-205 >lncRNA Zfyve19-204 >retained intron Zfyve19-210 >retained intron Zfyve19-209 >lncRNA Zfyve19-211 >retained intron Contigs < AL772264.8 AL929318.11 > Genes < Dnajc17-201protein coding < Ppp1r14d-201protein coding (Comprehensive set... < Dnajc17-202retained intron < Ppp1r14d-202protein coding Regulatory Build 119.21Mb 119.22Mb 119.23Mb Reverse strand 31.79 kb Regulation Legend Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000110820 < Ppp1r14d-202protein coding Reverse strand 11.73 kb ENSMUSP00000106... MobiDB lite Low complexity (Seg) Superfamily CPI-17 superfamily Pfam CPI-17 PANTHER CPI-17 PTHR16188:SF13 Gene3D CPI-17 superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant stop retained variant Scale bar 0 20 40 60 80 100 120 140 175 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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