https://www.alphaknockout.com Mouse Plekhg2 Knockout Project (CRISPR/Cas9) Objective: To create a Plekhg2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Plekhg2 gene (NCBI Reference Sequence: NM_138752 ; Ensembl: ENSMUSG00000037552 ) is located on Mouse chromosome 7. 19 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 19 (Transcript: ENSMUST00000094644). Exon 3~16 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 3 starts from about 2.73% of the coding region. Exon 3~16 covers 34.43% of the coding region. The size of effective KO region: ~6200 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 11 13 15 1 3 4 5 6 7 8 9 10 12 14 16 19 Legends Exon of mouse Plekhg2 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 617 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1298 bp section downstream of Exon 16 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(617bp) | A(25.77% 159) | C(19.12% 118) | T(21.39% 132) | G(33.71% 208) Note: The 617 bp section upstream of Exon 3 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(1298bp) | A(29.35% 381) | C(20.11% 261) | T(23.73% 308) | G(26.81% 348) Note: The 1298 bp section downstream of Exon 16 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 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 617 1 617 617 100.0% chr7 - 28370498 28371114 617 browser details YourSeq 24 320 347 617 85.2% chr15 + 58592031 58592057 27 browser details YourSeq 22 124 147 617 87.0% chr10 - 103873805 103873827 23 browser details YourSeq 22 278 303 617 84.0% chr14 + 42356176 42356200 25 browser details YourSeq 22 278 303 617 84.0% chr14 + 41216411 41216435 25 browser details YourSeq 21 450 474 617 92.0% chr16 - 21924961 21924985 25 browser details YourSeq 20 100 119 617 100.0% chr10 - 111361649 111361668 20 browser details YourSeq 20 231 250 617 100.0% chr13 + 37288190 37288209 20 Note: The 617 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 1298 1 1298 1298 100.0% chr7 - 28363000 28364297 1298 browser details YourSeq 218 443 737 1298 90.9% chr7 - 79356716 79357047 332 browser details YourSeq 204 434 741 1298 92.9% chr17 + 24523547 24523959 413 browser details YourSeq 190 451 744 1298 90.3% chr1 - 118434448 118434764 317 browser details YourSeq 189 458 728 1298 90.2% chr4 - 116873565 116874121 557 browser details YourSeq 189 442 744 1298 87.3% chr17 - 70806990 70807452 463 browser details YourSeq 189 437 755 1298 89.8% chr15 - 89327829 89328311 483 browser details YourSeq 188 442 728 1298 91.3% chr10 - 67258902 67259206 305 browser details YourSeq 187 439 741 1298 89.8% chr13 + 58359730 58360078 349 browser details YourSeq 184 432 728 1298 89.4% chr3 + 127550647 127550977 331 browser details YourSeq 183 442 731 1298 89.0% chr9 - 57056035 57056346 312 browser details YourSeq 182 423 740 1298 90.3% chr16 - 20503469 20504007 539 browser details YourSeq 181 484 742 1298 91.8% chr15 - 34445133 34445423 291 browser details YourSeq 170 427 741 1298 91.3% chr4 - 137621917 137622461 545 browser details YourSeq 168 441 728 1298 86.9% chr16 + 17881956 17882305 350 browser details YourSeq 160 384 646 1298 92.2% chr5 + 123411372 123411847 476 browser details YourSeq 157 460 730 1298 84.9% chr1 - 155546621 155547002 382 browser details YourSeq 153 441 911 1298 80.8% chr5 - 139250261 139250550 290 browser details YourSeq 153 460 753 1298 89.6% chr17 + 24465282 24553546 88265 browser details YourSeq 152 443 673 1298 91.0% chr8 - 25655989 25656241 253 Note: The 1298 bp section downstream of Exon 16 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Plekhg2 pleckstrin homology domain containing, family G (with RhoGef domain) member 2 [ Mus musculus (house mouse) ] Gene ID: 101497, updated on 3-Sep-2019 Gene summary Official Symbol Plekhg2 provided by MGI Official Full Name pleckstrin homology domain containing, family G (with RhoGef domain) member 2 provided by MGI Primary source MGI:MGI:2141874 See related Ensembl:ENSMUSG00000037552 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 Clg; Cslg; AI194308 Expression Broad expression in thymus adult (RPKM 36.3), spleen adult (RPKM 18.2) and 21 other tissuesS ee more Orthologs human all Genomic context Location: 7 A3; 7 16.71 cM See Plekhg2 in Genome Data Viewer Exon count: 20 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (28359603..28372681, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (29144623..29157681, complement) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Plekhg2 ENSMUSG00000037552 Description pleckstrin homology domain containing, family G (with RhoGef domain) member 2 [Source:MGI Symbol;Acc:MGI:2141874] Gene Synonyms Clg Location Chromosome 7: 28,359,604-28,372,599 reverse strand. GRCm38:CM001000.2 About this gene This gene has 10 transcripts (splice variants), 86 orthologues, 20 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Plekhg2- ENSMUST00000119990.7 4880 1340aa ENSMUSP00000112881.1 Protein coding CCDS52162 G5E8T4 TSL:1 202 GENCODE basic APPRIS ALT2 Plekhg2- ENSMUST00000094644.10 4623 1341aa ENSMUSP00000092228.4 Protein coding CCDS21040 E9QKB6 TSL:1 201 GENCODE basic APPRIS P3 Plekhg2- ENSMUST00000121085.7 4649 1365aa ENSMUSP00000113449.1 Protein coding - D3Z5N8 TSL:2 203 GENCODE basic APPRIS ALT2 Plekhg2- ENSMUST00000144700.7 3721 912aa ENSMUSP00000115651.1 Protein coding - A0A0R4J1S3 CDS 3' 206 incomplete TSL:1 Plekhg2- ENSMUST00000147362.7 729 205aa ENSMUSP00000118217.1 Protein coding - D3YY99 CDS 3' 207 incomplete TSL:3 Plekhg2- ENSMUST00000147887.1 476 63aa ENSMUSP00000122050.1 Protein coding - D3Z1B1 CDS 3' 209 incomplete TSL:3 Plekhg2- ENSMUST00000152281.1 858 56aa ENSMUSP00000117062.1 Nonsense mediated - D6RFC7 TSL:5 210 decay Plekhg2- ENSMUST00000128015.7 5511 No - Retained intron - - TSL:2 204 protein Plekhg2- ENSMUST00000129145.7 3074 No - Retained intron - - TSL:2 205 protein Plekhg2- ENSMUST00000147767.1 661 No - Retained intron - - TSL:3 208 protein Page 7 of 9 https://www.alphaknockout.com 33.00 kb Forward strand 28.35Mb 28.36Mb 28.37Mb 28.38Mb Genes Rps16-201 >protein coding Gm44710-201 >lncRNA (Comprehensive set... Rps16-202 >retained intron Rps16-203 >retained intron AF357399-201 >snoRNA Contigs < AC149606.6 Genes (Comprehensive set... < Plekhg2-201protein coding < Zfp36-201protein coding < Plekhg2-204retained intron < Zfp36-202protein coding < Plekhg2-202protein coding < Plekhg2-203protein coding < Plekhg2-205retained intron < Plekhg2-207protein coding < Plekhg2-206protein coding < Plekhg2-208retained intron < Plekhg2-210nonsense mediated decay < Plekhg2-209protein coding Regulatory Build 28.35Mb 28.36Mb 28.37Mb 28.38Mb Reverse strand 33.00 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000094644 < Plekhg2-201protein coding Reverse strand 12.63 kb ENSMUSP00000092... MobiDB lite Low complexity (Seg) Superfamily SSF50729 Dbl homology (DH) domain superfamily SMART Pleckstrin homology domain Dbl homology (DH) domain Pfam Pleckstrin homology domain Dbl homology (DH) domain PROSITE profiles Dbl homology (DH) domain Pleckstrin homology domain PANTHER PTHR45924:SF3 PTHR45924 Gene3D Dbl homology (DH) domain superfamily PH-like domain superfamily CDD cd13243 Dbl homology (DH) domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1341 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.