https://www.alphaknockout.com Mouse Ripor2 Knockout Project (CRISPR/Cas9) Objective: To create a Ripor2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ripor2 gene (NCBI Reference Sequence: NM_029679.2 ; Ensembl: ENSMUSG00000036006 ) is located on Mouse chromosome 13. 23 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 23 (Transcript: ENSMUST00000110384). Exon 2~7 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: Homozygous knockout mice are deaf. The gene product is expressed in the basal region of cochlear hair cell stereocillia, which are disorganized and malformed in null mice. Exon 2 starts from about 1.55% of the coding region. Exon 2~7 covers 18.24% of the coding region. The size of effective KO region: ~7914 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' 1 2 3 4 5 6 7 23 Legends Exon of mouse Ripor2 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 2000 bp section upstream of Exon 2 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 7 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(2000bp) | A(27.25% 545) | C(20.95% 419) | T(27.3% 546) | G(24.5% 490) Note: The 2000 bp section upstream of Exon 2 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(2000bp) | A(27.6% 552) | C(23.3% 466) | T(24.25% 485) | G(24.85% 497) Note: The 2000 bp section downstream of Exon 7 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 2000 1 2000 2000 100.0% chr13 + 24667961 24669960 2000 browser details YourSeq 258 19 369 2000 89.2% chr13 - 24666179 24666519 341 browser details YourSeq 101 1325 1436 2000 99.1% chr10 - 73797011 73797122 112 browser details YourSeq 88 1310 1437 2000 92.9% chr13 + 114922791 114922917 127 browser details YourSeq 77 1303 1405 2000 94.3% chr1 + 97778154 97778256 103 browser details YourSeq 74 1352 1435 2000 94.1% chr1 - 153668687 153668770 84 browser details YourSeq 65 1310 1409 2000 83.4% chr11 - 36018962 36019043 82 browser details YourSeq 54 1303 1378 2000 81.1% chr9 + 82688441 82688499 59 browser details YourSeq 54 76 159 2000 86.6% chr10 + 62361888 62361970 83 browser details YourSeq 50 41 146 2000 77.6% chr1 + 36695828 36695916 89 browser details YourSeq 46 1393 1438 2000 100.0% chr14 - 32777396 32777441 46 browser details YourSeq 42 127 260 2000 93.8% chr11 - 84442062 84463723 21662 browser details YourSeq 42 1301 1369 2000 76.4% chr13 + 33184422 33184484 63 browser details YourSeq 37 106 157 2000 97.5% chr7 - 112357105 112357185 81 browser details YourSeq 37 119 157 2000 92.2% chr13 + 95286150 95286187 38 browser details YourSeq 36 123 188 2000 82.2% chr7 - 112625051 112625117 67 browser details YourSeq 36 1303 1351 2000 87.5% chr11 - 100956611 100956657 47 browser details YourSeq 36 1403 1438 2000 100.0% chr15 + 25551530 25551565 36 browser details YourSeq 35 119 157 2000 89.5% chr18 + 14703972 14704009 38 browser details YourSeq 35 131 206 2000 92.7% chr15 + 84880654 84880730 77 Note: The 2000 bp section upstream of Exon 2 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 2000 1 2000 2000 100.0% chr13 + 24677875 24679874 2000 browser details YourSeq 107 378 1091 2000 86.3% chr11 - 99336187 99766684 430498 browser details YourSeq 89 358 453 2000 96.9% chr1 - 130576377 130576480 104 browser details YourSeq 85 379 952 2000 93.3% chr10 + 13884363 14012606 128244 browser details YourSeq 64 862 974 2000 84.8% chr7 + 113034395 113034505 111 browser details YourSeq 60 384 452 2000 94.3% chr12 + 18072645 18072714 70 browser details YourSeq 59 378 448 2000 96.9% chr10 - 35461214 35461289 76 browser details YourSeq 55 392 452 2000 96.8% chr7 - 43960529 43960608 80 browser details YourSeq 55 387 453 2000 88.4% chr1 + 97903527 97903589 63 browser details YourSeq 53 1024 1091 2000 89.8% chr6 + 128266830 128266898 69 browser details YourSeq 53 1027 1130 2000 72.3% chr10 + 43281953 43282029 77 browser details YourSeq 51 1028 1099 2000 86.2% chr2 + 169105954 169106042 89 browser details YourSeq 51 387 450 2000 84.3% chr12 + 51327496 51327553 58 browser details YourSeq 50 1024 1091 2000 84.9% chr11 - 4924285 4924351 67 browser details YourSeq 50 1030 1089 2000 91.7% chr14 + 87116992 87117051 60 browser details YourSeq 49 1024 1091 2000 86.6% chr11 + 80350341 80350409 69 browser details YourSeq 48 1024 1091 2000 85.3% chr5 - 146453015 146453082 68 browser details YourSeq 48 1026 1091 2000 86.4% chr12 - 55678017 55678082 66 browser details YourSeq 48 1026 1093 2000 85.3% chr10 - 31612225 31612292 68 browser details YourSeq 48 1028 1091 2000 87.5% chr1 + 133523290 133523353 64 Note: The 2000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Ripor2 RHO family interacting cell polarization regulator 2 [ Mus musculus (house mouse) ] Gene ID: 193385, updated on 26-Jun-2020 Gene summary Official Symbol Ripor2 provided by MGI Official Full Name RHO family interacting cell polarization regulator 2 provided by MGI Primary source MGI:MGI:2444879 See related Ensembl:ENSMUSG00000036006 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 Fam65b Expression Broad expression in spleen adult (RPKM 12.9), CNS E18 (RPKM 6.6) and 18 other tissues See more Orthologs human all Genomic context Location: 13; 13 A3.1 See Ripor2 in Genome Data Viewer Exon count: 30 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (24501524..24733806) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (24706479..24825675) Chromosome 13 - NC_000079.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 14 transcripts Gene: Ripor2 ENSMUSG00000036006 Description RHO family interacting cell polarization regulator 2 [Source:MGI Symbol;Acc:MGI:2444879] Gene Synonyms 1700108N18Rik, 6330500D04Rik, E430013J17Rik, Fam65b Location Chromosome 13: 24,501,525-24,733,816 forward strand. GRCm38:CM001006.2 About this gene This gene has 14 transcripts (splice variants), 280 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ripor2- ENSMUST00000110384.8 5608 1078aa ENSMUSP00000106013.2 Protein coding CCDS36622 Q80U16 TSL:1 205 GENCODE basic APPRIS ALT2 Ripor2- ENSMUST00000110383.7 5476 1053aa ENSMUSP00000106012.1 Protein coding CCDS70438 A6PW28 TSL:1 204 GENCODE basic APPRIS ALT2 Ripor2- ENSMUST00000038477.6 3371 672aa ENSMUSP00000043663.6 Protein coding CCDS36623 Q80U16 TSL:1 201 GENCODE basic APPRIS ALT2 Ripor2- ENSMUST00000091694.9 3142 633aa ENSMUSP00000089286.3 Protein coding CCDS26377 Q80U16 TSL:1 203 GENCODE basic APPRIS P3 Ripor2- ENSMUST00000058009.15 2550 389aa ENSMUSP00000051342.9 Protein coding - Q80U16 TSL:2 202 GENCODE basic APPRIS ALT2 Ripor2- ENSMUST00000238974.1 2364 639aa ENSMUSP00000159186.1 Protein coding - A0A5F8MPY8 GENCODE 214 basic APPRIS ALT2 Ripor2- ENSMUST00000132689.7 3261 40aa ENSMUSP00000115689.1 Nonsense mediated - F2Z3V4 TSL:1 206 decay Ripor2- ENSMUST00000134370.7 2342 No - Retained intron - - TSL:2 207 protein Ripor2- ENSMUST00000175986.1 2004 No - Retained intron - - TSL:1 209 protein Ripor2- ENSMUST00000138547.7 1350 No - Retained intron - - TSL:1 208 protein Ripor2- ENSMUST00000177489.1 760 No - Retained intron - - TSL:5 213 protein Ripor2- ENSMUST00000177298.7 665 No - Retained intron - - TSL:2 212 protein Ripor2- ENSMUST00000177174.1 649 No - Retained intron - - TSL:3 211 protein Ripor2- ENSMUST00000176303.1 546 No - Retained intron - - TSL:5 210 protein Page 7 of 9 https://www.alphaknockout.com 252.29 kb Forward strand 24.5Mb 24.6Mb 24.7Mb Genes (Comprehensive set... Ripor2-214 >protein coding Ripor2-213 >retained intron Ripor2-204 >protein coding Ripor2-203 >protein coding Armh2-201 >protein coding Ripor2-207 >retained intron Ripor2-206 >nonsense mediated decay Ripor2-205 >protein coding Ripor2-212 >retained intronRipor2-211 >retained intron Ripor2-202 >protein coding Ripor2-201 >protein coding Ripor2-208 >retained intron Ripor2-210 >retained intron Ripor2-209 >retained intron Contigs AL589744.18 > AL591851.12 > AL513014.9 > AL589699.4 > Genes < Gm11346-204processed transcript (Comprehensive set..