https://www.alphaknockout.com Mouse Glra3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Glra3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Glra3 gene (NCBI Reference Sequence: NM_080438 ; Ensembl: ENSMUSG00000038257 ) is located on Mouse chromosome 8. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000000275). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Glra3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-365J12 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: Homozygous null mice are fertile and display decreased inflammatory pain sensitization without any gross abnormalities in the brain or spinal cord. Exon 3 starts from about 17.22% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 13863 bp, and the size of intron 3 for 3'-loxP site insertion: 34397 bp. The size of effective cKO region: ~568 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 3 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Glra3 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(7068bp) | A(32.5% 2297) | C(18.35% 1297) | T(32.41% 2291) | G(16.74% 1183) 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% chr8 + 56001868 56004867 3000 browser details YourSeq 242 328 671 3000 87.5% chrX - 164589226 164589587 362 browser details YourSeq 236 326 669 3000 85.4% chr2 - 71491597 71491947 351 browser details YourSeq 236 333 671 3000 86.2% chr15 - 92932598 92932944 347 browser details YourSeq 236 326 671 3000 85.6% chr1 - 189296523 189296884 362 browser details YourSeq 233 322 671 3000 85.7% chr8 - 124632552 124632898 347 browser details YourSeq 229 333 679 3000 85.7% chr1 + 24156126 24156477 352 browser details YourSeq 228 317 671 3000 87.0% chr5 + 45424521 45424901 381 browser details YourSeq 226 333 671 3000 85.6% chr16 + 52658669 52659015 347 browser details YourSeq 225 319 670 3000 84.6% chr3 + 122354841 122355201 361 browser details YourSeq 222 341 671 3000 86.9% chr3 - 123252867 123253211 345 browser details YourSeq 220 334 671 3000 85.1% chr6 + 7761991 7762340 350 browser details YourSeq 219 319 669 3000 85.9% chr1 - 129448464 129448820 357 browser details YourSeq 217 333 671 3000 83.5% chr14 + 74471650 74471995 346 browser details YourSeq 216 333 667 3000 86.9% chr13 + 47305609 47305942 334 browser details YourSeq 215 325 671 3000 85.5% chr4 - 74621226 74621594 369 browser details YourSeq 215 326 671 3000 84.1% chr14 - 119654031 119654396 366 browser details YourSeq 215 329 671 3000 89.2% chr19 + 22052321 22052667 347 browser details YourSeq 214 320 670 3000 84.6% chrX - 162536386 162536737 352 browser details YourSeq 214 319 669 3000 86.2% chr16 + 33642863 33643230 368 Note: The 3000 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 3000 1 3000 3000 100.0% chr8 + 56005436 56008435 3000 browser details YourSeq 210 843 1502 3000 90.4% chr17 - 55350229 55350884 656 browser details YourSeq 206 1281 1516 3000 94.4% chr8 + 78673691 78673929 239 browser details YourSeq 204 1281 1516 3000 94.0% chr17 - 74901465 74901702 238 browser details YourSeq 203 1281 1516 3000 94.0% chr4 - 16424945 16425182 238 browser details YourSeq 201 1227 1502 3000 92.0% chr12 + 119542618 119543044 427 browser details YourSeq 198 1281 1502 3000 94.1% chr8 - 21819086 21819306 221 browser details YourSeq 197 1281 1505 3000 93.8% chr3 - 81553450 81553674 225 browser details YourSeq 197 1281 1505 3000 93.3% chr12 - 118037404 118037627 224 browser details YourSeq 196 1281 1502 3000 93.7% chrX - 157514859 157515079 221 browser details YourSeq 196 1281 1502 3000 93.7% chr9 - 11715269 11715489 221 browser details YourSeq 196 1283 1502 3000 94.1% chr1 - 177183551 177183769 219 browser details YourSeq 195 1281 1502 3000 93.7% chr3 - 82919236 82919456 221 browser details YourSeq 195 1288 1637 3000 89.8% chr10 - 105902839 105903339 501 browser details YourSeq 195 1281 1502 3000 93.2% chr14 + 74265278 74265498 221 browser details YourSeq 194 1281 1502 3000 93.2% chr6 - 101577935 101578155 221 browser details YourSeq 194 1281 1502 3000 93.7% chr5 - 116458271 116458492 222 browser details YourSeq 194 1281 1502 3000 93.7% chr13 - 16956364 16956585 222 browser details YourSeq 194 1281 1507 3000 93.4% chr1 - 59661991 59662217 227 browser details YourSeq 194 1281 1502 3000 93.2% chr1 - 58547495 58547715 221 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Glra3 glycine receptor, alpha 3 subunit [ Mus musculus (house mouse) ] Gene ID: 110304, updated on 15-Aug-2019 Gene summary Official Symbol Glra3 provided by MGI Official Full Name glycine receptor, alpha 3 subunit provided by MGI Primary source MGI:MGI:95749 See related Ensembl:ENSMUSG00000038257 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 Expression Low expression observed in reference dataset See more Orthologs human all Genomic context Location: 8 B2; 8 29.69 cM See Glra3 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (55940418..56130070) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (58419622..58604149) Chromosome 8 - NC_000074.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Glra3 ENSMUSG00000038257 Description glycine receptor, alpha 3 subunit [Source:MGI Symbol;Acc:MGI:95749] Location Chromosome 8: 55,940,460-56,130,070 forward strand. GRCm38:CM001001.2 About this gene This gene has 2 transcripts (splice variants), 196 orthologues, 41 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Glra3-201 ENSMUST00000000275.8 6508 480aa ENSMUSP00000000275.7 Protein coding CCDS40340 G5E811 TSL:1 GENCODE basic APPRIS P1 Glra3-202 ENSMUST00000164020.1 3586 No protein - lncRNA - - TSL:1 209.61 kb Forward strand 55.95Mb 56.00Mb 56.05Mb 56.10Mb Genes (Comprehensive set... Glra3-202 >lncRNA Glra3-201 >protein coding Contigs < AC117729.9 < AC119900.7 Genes < Gm7351-201processed pseudogene< Gm45366-201processed pseudogene (Comprehensive set... Regulatory Build 55.95Mb 56.00Mb 56.05Mb 56.10Mb Reverse strand 209.61 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding pseudogene RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000000275 189.56 kb Forward strand Glra3-201 >protein coding ENSMUSP00000000... Transmembrane heli... Low complexity (Seg) TIGRFAM Neurotransmitter-gated ion-channel Superfamily Neurotransmitter-gated ion-channel ligand-binding domain superfamily Neurotransmitter-gated ion-channel transmembrane domain superfamily Prints Glycine receptor alpha3 Gamma-aminobutyric acid A receptor/Glycine receptor alpha Neurotransmitter-gated ion-channel Glycine receptor alpha Pfam Neurotransmitter-gated ion-channel transmembrane domain Neurotransmitter-gated ion-channel ligand-binding domain PROSITE patterns Neurotransmitter-gated ion-channel, conserved site PANTHER Neurotransmitter-gated ion-channel PTHR18945:SF455 Gene3D Neurotransmitter-gated ion-channel ligand-binding domain superfamily CDD cd19009 cd19060 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 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.