https://www.alphaknockout.com Mouse Pcdhb5 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pcdhb5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pcdhb5 gene (NCBI Reference Sequence: NM_053130 ; Ensembl: ENSMUSG00000063687 ) is located on Mouse chromosome 18. 1 exon is identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 1 (Transcript: ENSMUST00000078271). 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 Pcdhb5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-480H17 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 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~2409 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region gRNA region Wildtype allele A T A T 5' G A 3' 1 Targeting vector A T A T G A Targeted allele A T A T G A Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Pcdhb5 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. 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(8376bp) | A(29.63% 2482) | C(19.72% 1652) | T(29.43% 2465) | G(21.22% 1777) 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% chr18 + 37317570 37320569 3000 browser details YourSeq 182 739 1442 3000 87.7% chr5 + 64620042 64620682 641 browser details YourSeq 151 697 1013 3000 86.9% chr8 - 61749419 61749747 329 browser details YourSeq 142 673 1001 3000 82.5% chr2 + 169924932 169925229 298 browser details YourSeq 142 734 1046 3000 84.4% chr16 + 33603899 33604183 285 browser details YourSeq 141 1331 1494 3000 93.3% chr12 + 20827766 20827934 169 browser details YourSeq 140 669 1009 3000 86.6% chr17 - 6880840 6881194 355 browser details YourSeq 138 1331 1481 3000 96.1% chr13 + 90901416 90901567 152 browser details YourSeq 137 749 1011 3000 89.1% chr10 + 120837707 120837970 264 browser details YourSeq 136 736 1023 3000 91.6% chr9 + 105794346 105794633 288 browser details YourSeq 135 716 1012 3000 91.6% chr10 - 126447206 126447508 303 browser details YourSeq 134 721 955 3000 86.5% chr2 + 159183964 159184210 247 browser details YourSeq 131 1331 1481 3000 93.4% chr8 - 111144505 111144655 151 browser details YourSeq 131 732 1046 3000 85.1% chr17 - 26477438 26477743 306 browser details YourSeq 131 723 1046 3000 88.0% chr11 - 115613792 115620069 6278 browser details YourSeq 131 652 1010 3000 84.6% chr10 - 126025431 126025787 357 browser details YourSeq 130 660 950 3000 85.4% chr4 - 47792845 47793138 294 browser details YourSeq 130 732 1029 3000 86.5% chrX + 94034345 94034849 505 browser details YourSeq 130 1334 1481 3000 94.0% chr8 + 106040818 106040965 148 browser details YourSeq 129 748 1004 3000 89.3% chr5 - 118350469 118350731 263 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 + 37322946 37325945 3000 browser details YourSeq 532 1681 2753 3000 89.2% chr18 + 37345902 37346986 1085 browser details YourSeq 248 392 723 3000 88.5% chr9 + 80082766 80083084 319 browser details YourSeq 246 392 730 3000 88.6% chr13 - 96259829 96299984 40156 browser details YourSeq 242 339 723 3000 87.9% chr5 - 28549980 28550461 482 browser details YourSeq 239 387 722 3000 87.7% chrX - 95810679 95811004 326 browser details YourSeq 238 392 709 3000 89.6% chr13 - 93227599 93227912 314 browser details YourSeq 237 392 720 3000 86.7% chr15 + 3454457 3454773 317 browser details YourSeq 236 392 728 3000 85.8% chr8 + 91972162 91972470 309 browser details YourSeq 235 389 721 3000 87.2% chr10 + 115522709 115523036 328 browser details YourSeq 233 392 733 3000 88.9% chr10 + 29616191 29948690 332500 browser details YourSeq 232 392 723 3000 87.9% chr9 - 49093142 49093461 320 browser details YourSeq 231 392 722 3000 87.4% chr10 + 23094031 23094350 320 browser details YourSeq 230 392 723 3000 85.9% chr8 - 42833500 42833817 318 browser details YourSeq 230 389 709 3000 86.3% chr14 + 120448741 120449044 304 browser details YourSeq 229 392 722 3000 85.5% chr7 - 101166119 101166436 318 browser details YourSeq 229 392 723 3000 85.3% chr2 - 136652877 136653190 314 browser details YourSeq 229 392 722 3000 87.4% chr12 - 98212207 98212525 319 browser details YourSeq 229 392 722 3000 86.4% chr14 + 63324270 63324586 317 browser details YourSeq 229 389 723 3000 85.4% chr10 + 21509092 21509416 325 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Pcdhb5 protocadherin beta 5 [ Mus musculus (house mouse) ] Gene ID: 93876, updated on 11-Sep-2019 Gene summary Official Symbol Pcdhb5 provided by MGI Official Full Name protocadherin beta 5 provided by MGI Primary source MGI:MGI:2136739 See related Ensembl:ENSMUSG00000063687 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 PcdhbE; Pcdhb4A Genomic context Location: 18; 18 B3 See Pcdhb5 in Genome Data Viewer Exon count: 1 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (37320381..37323913) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (37480035..37483567) Chromosome 18 - NC_000084.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Pcdhb5 ENSMUSG00000063687 Description protocadherin beta 5 [Source:MGI Symbol;Acc:MGI:2136739] Gene Synonyms Pcdhb4A, PcdhbE Location Chromosome 18: 37,320,381-37,323,915 forward strand. GRCm38:CM001011.2 About this gene This gene has 1 transcript (splice variant), 79 orthologues, 69 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pcdhb5-201 ENSMUST00000078271.3 3535 792aa ENSMUSP00000077389.2 Protein coding CCDS29173 Q91XZ5 TSL:NA GENCODE basic APPRIS P1 23.54 kb Forward strand 37.315Mb 37.320Mb 37.325Mb 37.330Mb Genes Gm37388-201 >protein coding (Comprehensive set... Gm42416-201 >protein coding Pcdhb4-201 >protein coding Pcdhb5-201 >protein coding Gm37013-201 >protein coding Contigs AC020974.4 > Regulatory Build 37.315Mb 37.320Mb 37.325Mb 37.330Mb Reverse strand 23.54 kb Regulation Legend CTCF Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000078271 3.54 kb Forward strand Pcdhb5-201 >protein coding ENSMUSP00000077... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Cadherin-like superfamily SMART Cadherin-like Prints Cadherin-like Pfam Cadherin, N-terminal Cadherin, cytoplasmic C-terminal domain Cadherin-like PROSITE profiles PS50268 PROSITE patterns Cadherin conserved site PANTHER Protocadherin beta-4 PTHR24028 Gene3D 2.60.40.60 CDD cd11304 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 792 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.