https://www.alphaknockout.com Mouse Grip2 Knockout Project (CRISPR/Cas9) Objective: To create a Grip2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Grip2 gene (NCBI Reference Sequence: NM_001159507 ; Ensembl: ENSMUSG00000030098 ) is located on Mouse chromosome 6. 24 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 24 (Transcript: ENSMUST00000162300). Exon 2~12 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 null mice are born in numbers expected by the Mendelian ratio and show no overt phenotype. Exon 2 starts from about 1.79% of the coding region. Exon 2~12 covers 46.19% of the coding region. The size of effective KO region: ~8114 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 8 9 10 11 12 24 Legends Exon of mouse Grip2 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 361 bp section downstream of Exon 12 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. 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(25.85% 517) | C(21.8% 436) | T(26.15% 523) | G(26.2% 524) 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(361bp) | A(24.1% 87) | C(27.7% 100) | T(24.38% 88) | G(23.82% 86) Note: The 361 bp section downstream of Exon 12 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% chr6 - 91788445 91790444 2000 browser details YourSeq 40 1080 1245 2000 95.5% chr3 + 116416038 116416218 181 browser details YourSeq 38 1695 1743 2000 93.2% chr12 - 102557721 102557771 51 browser details YourSeq 38 1695 1761 2000 95.3% chr10 + 81586065 81586185 121 browser details YourSeq 35 1153 1245 2000 70.8% chr4 - 129127066 129127137 72 browser details YourSeq 35 1312 1557 2000 83.4% chr17 - 65368164 65368407 244 browser details YourSeq 35 1068 1107 2000 97.4% chr11 - 96220569 96220801 233 browser details YourSeq 30 86 115 2000 100.0% chr14 - 28458571 28458600 30 browser details YourSeq 29 86 116 2000 96.8% chr15 - 98803901 98803931 31 browser details YourSeq 29 1693 1737 2000 96.8% chr8 + 4194438 4194483 46 browser details YourSeq 29 1694 1744 2000 91.5% chr5 + 120954813 120954865 53 browser details YourSeq 29 1179 1229 2000 75.8% chr3 + 158399000 158399044 45 browser details YourSeq 29 1206 1245 2000 91.5% chr3 + 66526226 66526266 41 browser details YourSeq 29 1065 1097 2000 87.5% chr13 + 40563036 40563067 32 browser details YourSeq 28 86 117 2000 93.8% chr5 - 33685303 33685334 32 browser details YourSeq 27 1694 1737 2000 96.6% chr5 - 113279826 113279871 46 browser details YourSeq 27 1153 1191 2000 84.7% chr11 - 59152791 59152829 39 browser details YourSeq 27 88 116 2000 96.6% chr2 + 32100060 32100088 29 browser details YourSeq 27 1076 1104 2000 96.6% chr15 + 18764208 18764236 29 browser details YourSeq 26 1064 1097 2000 88.3% chr13 - 58595335 58595368 34 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 361 1 361 361 100.0% chr6 - 91779970 91780330 361 browser details YourSeq 27 98 125 361 100.0% chr3 - 75920306 75920795 490 browser details YourSeq 23 168 194 361 79.2% chr1 - 75364410 75364433 24 browser details YourSeq 22 178 201 361 87.0% chr1 + 178794441 178794463 23 browser details YourSeq 21 66 88 361 95.7% chr12 - 119539631 119539653 23 browser details YourSeq 21 76 100 361 92.0% chr6 + 97718381 97718405 25 browser details YourSeq 21 47 68 361 100.0% chr11 + 47504331 47504353 23 browser details YourSeq 20 308 327 361 100.0% chr14 - 67031969 67031988 20 browser details YourSeq 20 220 239 361 100.0% chr1 + 172314244 172314263 20 Note: The 361 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Grip2 glutamate receptor interacting protein 2 [ Mus musculus (house mouse) ] Gene ID: 243547, updated on 12-Aug-2019 Gene summary Official Symbol Grip2 provided by MGI Official Full Name glutamate receptor interacting protein 2 provided by MGI Primary source MGI:MGI:2681173 See related Ensembl:ENSMUSG00000030098 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 Broad expression in frontal lobe adult (RPKM 10.3), CNS E18 (RPKM 10.0) and 18 other tissues See more Orthologs human all Genomic context Location: 6; 6 D1 See Grip2 in Genome Data Viewer Exon count: 27 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (91761452..91843954, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (91711503..91757387, complement) Chromosome 6 - NC_000072.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Grip2 ENSMUSG00000030098 Description glutamate receptor interacting protein 2 [Source:MGI Symbol;Acc:MGI:2681173] Location Chromosome 6: 91,761,509-91,827,250 reverse strand. GRCm38:CM000999.2 About this gene This gene has 6 transcripts (splice variants), 262 orthologues, 6 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Grip2- ENSMUST00000162300.7 5206 1042aa ENSMUSP00000124709.1 Protein coding CCDS51852 G3XA20 TSL:5 205 GENCODE basic APPRIS P2 Grip2- ENSMUST00000159684.7 3006 1001aa ENSMUSP00000125047.1 Protein coding - E0CXS4 TSL:5 201 GENCODE basic APPRIS ALT2 Grip2- ENSMUST00000162293.7 5520 821aa ENSMUSP00000124717.1 Nonsense mediated - E0CXE0 TSL:5 204 decay Grip2- ENSMUST00000161566.7 1008 174aa ENSMUSP00000123941.1 Nonsense mediated - E0CYS1 TSL:5 203 decay Grip2- ENSMUST00000161545.1 763 119aa ENSMUSP00000125538.1 Nonsense mediated - F7BQ55 CDS 5' 202 decay incomplete TSL:3 Grip2- ENSMUST00000205964.1 3555 No - Retained intron - - TSL:NA 206 protein Page 7 of 9 https://www.alphaknockout.com 85.74 kb Forward strand 91.76Mb 91.78Mb 91.80Mb 91.82Mb Genes Slc6a6-201 >protein coding (Comprehensive set... Slc6a6-211 >retained intron Contigs < AC118245.17 AC162914.2 > Genes (Comprehensive set... < Grip2-204nonsense mediated decay < Grip2-205protein coding < Grip2-201protein coding < Grip2-203nonsense mediated decay < Grip2-202nonsense mediated decay < Grip2-206retained intron Regulatory Build 91.76Mb 91.78Mb 91.80Mb 91.82Mb Reverse strand 85.74 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000162300 < Grip2-205protein coding Reverse strand 45.95 kb ENSMUSP00000124... MobiDB lite Low complexity (Seg) Superfamily PDZ superfamily SMART PDZ domain Pfam PDZ domain PDZ domain 6 PROSITE profiles PDZ domain PANTHER Glutamate receptor-interacting protein 2 PTHR46227 Gene3D 2.30.42.10 CDD cd00992 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1042 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.