https://www.alphaknockout.com Mouse Pgm1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pgm1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pgm1 gene (NCBI Reference Sequence: None ; Ensembl: ENSMUSG00000025791 ) is located on Mouse chromosome 5. 11 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000102783). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pgm1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-283K16 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 4 starts from about 35.11% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 1327 bp, and the size of intron 5 for 3'-loxP site insertion: 2193 bp. The size of effective cKO region: ~1848 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' 5 1 9 2 3 8 4 7 6 5 4 6 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pgm1 Exon of mouse Hnrnpd 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. 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(8348bp) | A(34.42% 2873) | C(20.81% 1737) | T(29.24% 2441) | G(15.54% 1297) 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% chr5 + 99960269 99963268 3000 browser details YourSeq 134 1575 2038 3000 89.2% chr5 + 100035390 100035832 443 browser details YourSeq 24 1288 1316 3000 81.5% chr19 + 7440405 7440431 27 Note: The 3000 bp section upstream of Exon 4 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% chr5 + 99965117 99968116 3000 browser details YourSeq 175 1029 2272 3000 83.5% chrX + 162845828 162846036 209 browser details YourSeq 153 1230 1419 3000 88.6% chr4 - 150183022 150183199 178 browser details YourSeq 153 1242 1424 3000 92.8% chr3 + 71657980 71658164 185 browser details YourSeq 149 1246 1430 3000 90.9% chr8 + 126914116 126914299 184 browser details YourSeq 148 1237 1427 3000 86.2% chr13 - 14041591 14041772 182 browser details YourSeq 139 1255 1419 3000 89.2% chrX - 52255919 52256075 157 browser details YourSeq 138 1234 1424 3000 86.9% chr13 - 55173541 55173722 182 browser details YourSeq 138 1246 1410 3000 89.9% chr12 + 83608802 83608960 159 browser details YourSeq 137 1246 1414 3000 88.7% chr7 + 127265205 127265366 162 browser details YourSeq 137 1249 1411 3000 89.9% chr1 + 164414245 164414403 159 browser details YourSeq 136 1232 1411 3000 87.6% chr3 - 150861202 150861374 173 browser details YourSeq 136 1244 1412 3000 89.3% chr2 + 155530054 155530218 165 browser details YourSeq 135 1246 1414 3000 90.8% chr7 - 22059364 22059533 170 browser details YourSeq 135 1246 1414 3000 87.6% chr15 + 52211045 52211206 162 browser details YourSeq 132 1246 1423 3000 84.8% chr11 - 26421138 26421304 167 browser details YourSeq 132 1246 1424 3000 93.0% chr1 - 142534884 142535064 181 browser details YourSeq 132 1255 1424 3000 87.3% chr8 + 123051152 123051318 167 browser details YourSeq 132 1249 1414 3000 87.5% chr1 + 81036634 81036795 162 browser details YourSeq 131 1245 1407 3000 90.4% chr5 + 122116550 122116711 162 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Pgm1 phosphoglucomutase 1 [ Mus musculus (house mouse) ] Gene ID: 72157, updated on 10-Oct-2019 Gene summary Official Symbol Pgm1 provided by MGI Official Full Name phosphoglucomutase 1 provided by MGI Primary source MGI:MGI:97565 See related Ensembl:ENSMUSG00000025791 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 Pgm2; Pgm-2; Pgm1a; AA407108; AI098105; 2610020G18Rik Annotation Note: The name of this locus, Pgm1, was recently updated by Mouse Genomic Informatics to be consistent with information mammalian orthologs identified as Pgm1. Note that this gene is frequently referred to as Pgm2 in the literature. [02 Oct Expression 2019] Orthologs Ubiquitous expression in liver E18 (RPKM 86.8), mammary gland adult (RPKM 70.6) and 27 other tissues See more human all Genomic context Location: 4 C6; 4 45.71 cM See Pgm1 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (99929451..99987294) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (99602056..99659899) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Pgm1 ENSMUSG00000025791 Description phosphoglucomutase 1 [Source:MGI Symbol;Acc:MGI:97565] Gene Synonyms 2610020G18Rik, Pgm-2, Pgm1a, Pgm2 Location Chromosome 4: 99,929,414-99,987,294 forward strand. GRCm38:CM000997.2 View alleles of this gene on alternative sequences About this gene This gene has 2 transcripts (splice variants), 1 gene allele, 198 orthologues, 5 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pgm1-201 ENSMUST00000058351.15 2371 562aa ENSMUSP00000061227.9 Protein coding CCDS18388 Q9D0F9 TSL:1 GENCODE basic APPRIS P2 Pgm1-202 ENSMUST00000102783.4 2523 580aa ENSMUSP00000099844.4 Protein coding - A2CEK3 TSL:1 GENCODE basic APPRIS ALT1 77.88 kb Forward strand Genes (Comprehensive set... Pgm1-201 >protein coding Pgm1-202 >protein coding Contigs CR536609.3 > Genes < Itgb3bp-203lncRNA (Comprehensive set... Regulatory Build Reverse strand 77.88 kb Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000102783 31.55 kb Forward strand Pgm1-202 >protein coding ENSMUSP00000099... Low complexity (Seg) Superfamily Alpha-D-phosphohexomutase, C-terminal domain superfamily Alpha-D-phosphohexomutase, alpha/beta/alpha I/II/III Prints Alpha-D-phosphohexomutase superfamily Pfam Alpha-D-phosphohexomutase, alpha/beta/alpha domain I Alpha-D-phosphohexomutase, alpha/beta/alpha domain III Alpha-D-phosphohexomutase, alpha/beta/alpha domain II Alpha-D-phosphohexomutase, C-terminal PROSITE patterns Alpha-D-phosphohexomutase, conserved site PANTHER PTHR22573 PTHR22573:SF55 Gene3D 3.40.120.10 3.30.310.50 CDD cd03085 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 580 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.