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Mouse Pcdhga11 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Pcdhga11 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pcdhga11 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pcdhga11 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pcdhga11 gene (NCBI Reference Sequence: NM_033594 ; Ensembl: ENSMUSG00000102742 ) is located on Mouse chromosome 18. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000061279). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pcdhga11 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-56B11 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 1 covers 86.71% of the coding region. Start codon is in exon 1, and stop codon is in exon 4. The size of intron 1 for 3'-loxP site insertion: 66653 bp. The size of effective cKO region: ~2687 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele A T 5' G gRNA region 3' 1 1 4 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pcdhgb7 Homology arm Exon of mouse Pcdhga11 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. 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(8927bp) | A(24.7% 2205) | C(23.74% 2119) | T(30.1% 2687) | G(21.46% 1916) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chr18 + 37752942 37755941 3000 browser details YourSeq 662 23 1094 3000 83.4% chr18 + 37681613 37733404 51792 browser details YourSeq 655 17 1098 3000 80.4% chr18 + 37732327 37744507 12181 browser details YourSeq 592 44 940 3000 83.1% chr18 + 37763094 37763991 898 browser details YourSeq 485 273 942 3000 87.1% chr18 + 37681863 37682532 670 browser details YourSeq 458 142 942 3000 83.0% chr18 + 37691265 37722658 31394 browser details YourSeq 147 1337 1792 3000 84.8% chr16 - 13645319 13645723 405 browser details YourSeq 129 1687 2249 3000 82.8% chr15 + 99782612 99783004 393 browser details YourSeq 126 1689 2246 3000 82.9% chr2 - 104693791 104694213 423 browser details YourSeq 123 1691 2260 3000 81.0% chr5 + 147821562 147821828 267 browser details YourSeq 117 1692 2252 3000 80.8% chr9 + 110595819 110596177 359 browser details YourSeq 114 1430 1794 3000 81.4% chr17 - 29001384 29001582 199 browser details YourSeq 108 1692 2287 3000 86.8% chr2 + 23115632 23116199 568 browser details YourSeq 102 1327 1794 3000 86.8% chr8 - 111846219 111846662 444 browser details YourSeq 97 1673 1800 3000 90.9% chr10 + 81171428 81171577 150 browser details YourSeq 96 1689 1794 3000 96.3% chr5 + 14062356 14062483 128 browser details YourSeq 93 1360 1750 3000 93.5% chrX - 124232460 124233018 559 browser details YourSeq 93 1346 1794 3000 78.7% chr19 - 43498331 43498460 130 browser details YourSeq 92 1673 1793 3000 89.1% chr10 + 34636015 34636157 143 browser details YourSeq 91 1688 1792 3000 94.3% chr4 - 155945736 155945862 127 Note: The 3000 bp section upstream of Exon 1 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% chr18 + 37758619 37761618 3000 browser details YourSeq 339 2193 2961 3000 94.4% chr2 - 55622290 55622921 632 browser details YourSeq 336 2193 2567 3000 94.7% chrX - 108115895 108116246 352 browser details YourSeq 335 2163 2523 3000 98.6% chr2 - 3211866 3212290 425 browser details YourSeq 335 2193 2536 3000 98.9% chr8 + 32720418 32720777 360 browser details YourSeq 334 2193 2535 3000 98.9% chr8 - 30705387 30705736 350 browser details YourSeq 333 2193 2534 3000 98.9% chrX - 113721519 113721861 343 browser details YourSeq 333 2193 2623 3000 93.8% chr12 - 44731085 44731441 357 browser details YourSeq 333 2193 2534 3000 98.9% chrY + 19103807 19104149 343 browser details YourSeq 333 2193 2537 3000 98.6% chr5 + 22462543 22462895 353 browser details YourSeq 332 2193 2535 3000 98.6% chr2 - 129448167 129448511 345 browser details YourSeq 332 2193 2534 3000 98.9% chr19 - 43405885 43406241 357 browser details YourSeq 332 2193 2536 3000 98.6% chr9 + 7247368 7247711 344 browser details YourSeq 332 2193 2533 3000 98.9% chr2 + 110700028 110700369 342 browser details YourSeq 332 2193 2533 3000 98.9% chr13 + 78000796 78001137 342 browser details YourSeq 332 2193 2534 3000 98.9% chr1 + 62458203 62458549 347 browser details YourSeq 331 2193 2534 3000 98.6% chrX - 143256485 143256828 344 browser details YourSeq 331 2193 2534 3000 98.6% chrX - 97960195 97960538 344 browser details YourSeq 331 2193 2530 3000 99.2% chr6 - 117418864 117419204 341 browser details YourSeq 331 2193 2533 3000 98.6% chr3 - 93059101 93059441 341 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Pcdhga11 protocadherin gamma subfamily A, 11 [ Mus musculus (house mouse) ] Gene ID: 93723, updated on 12-Aug-2019 Gene summary Official Symbol Pcdhga11 provided by MGI Official Full Name protocadherin gamma subfamily A, 11 provided by MGI Primary source MGI:MGI:1935228 See related Ensembl:ENSMUSG00000102742 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Expression Broad expression in frontal lobe adult (RPKM 49.6), whole brain E14.5 (RPKM 45.5) and 24 other tissues See more Genomic context Location: 18; 18 B3 See Pcdhga11 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (37755773..37841873) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (37915427..38001527) Chromosome 18 - NC_000084.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Pcdhga11 ENSMUSG00000102742 Description protocadherin gamma subfamily A, 11 [Source:MGI Symbol;Acc:MGI:1935228] Location Chromosome 18: 37,755,731-37,841,873 forward strand. GRCm38:CM001011.2 About this gene This gene has 1 transcript (splice variant), 25 orthologues, 69 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pcdhga11-201 ENSMUST00000061279.9 4756 933aa ENSMUSP00000058362.7 Protein coding CCDS37784 Q91XY8 TSL:1 GENCODE basic APPRIS P1 106.14 kb Forward strand 37.76Mb 37.78Mb 37.80Mb 37.82Mb 37.84Mb Genes Pcdhga9-201 >protein coding (Comprehensive set... Pcdhgb4-202 >protein coding Pcdhgb1-201 >protein coding Pcdhga2-201 >protein coding Pcdhga7-201 >protein coding Pcdhgb6-201 >protein coding Pcdhga5-201 >protein coding Gm37388-201 >protein coding Pcdhgb2-202 >protein coding Pcdhga8-201 >protein coding Pcdhga3-201 >protein coding Pcdhgb5-201 >protein coding Gm37013-201 >protein coding Pcdhga6-201 >protein coding Gm42416-201 >protein coding Pcdhga4-201 >protein coding Pcdhga1-201 >protein coding Pcdhga10-201 >protein coding Pcdhgb7-201 >protein coding Pcdhga11-201 >protein coding Pcdhgb8-201 >polymorphic pseudogene Pcdhgb8-202 >protein coding Page 6 of 8 Pcdhga12-201 >protein coding Pcdhga12-202 >retained intron Pcdhgc3-203 >retained intron Gm38182-203 >lncRNA Pcdhgc3-201 >protein coding Pcdhgc3-202 >nonsense mediated decay Pcdhgc4-201 >protein coding Pcdhgc4-203 >protein coding Pcdhgc4-202 >retained intron Pcdhgc5-201 >protein coding Pcdhgc5-203 >protein coding Pcdhgc5-204 >protein coding Pcdhgc5-202 >retained intron Gm38182-201 >lncRNA Gm38182-202 >lncRNA Gm38182-204 >lncRNA Gm38182-205 >lncRNA Contigs AC020971.3 > AC129315.3 > Genes < Gm26672-202lncRNA < BC037039-201lncRNA < Gm29994-201lncRNA (Comprehensive set... < Gm26672-203lncRNA < Diaph1-205protein coding < Gm26672-201lncRNA < Diaph1-204protein coding < Gm26672-204retained intron < Diaph1-201protein coding < Diaph1-203protein coding < Diaph1-202protein coding < Diaph1-207lncRNA Regulatory Build 37.76Mb 37.78Mb 37.80Mb 37.82Mb 37.84Mb Reverse strand 106.14 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript pseudogene RNA gene 106.14 kb Forward strand 37.76Mb 37.78Mb 37.80Mb 37.82Mb
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