https://www.alphaknockout.com Mouse Nrip2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Nrip2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nrip2 gene (NCBI Reference Sequence: NM_021717 ; Ensembl: ENSMUSG00000001520 ) is located on Mouse chromosome 6. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000001561). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nrip2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-359I11 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 2 starts from about 27.66% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 4633 bp, and the size of intron 3 for 3'-loxP site insertion: 781 bp. The size of effective cKO region: ~2138 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Nrip2 Homology arm cKO region loxP site Page 2 of 8 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(8638bp) | A(25.69% 2219) | C(24.54% 2120) | T(25.8% 2229) | G(23.96% 2070) 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 8 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% chr6 + 128401686 128404685 3000 browser details YourSeq 127 1887 2349 3000 79.2% chr10 + 42428797 42429187 391 browser details YourSeq 125 944 1253 3000 83.1% chr4 - 123575354 123575633 280 browser details YourSeq 120 984 1238 3000 89.4% chr2 - 156866337 156890792 24456 browser details YourSeq 114 947 1248 3000 85.9% chr11 - 101490709 101792646 301938 browser details YourSeq 105 992 1249 3000 90.0% chr2 + 163762466 163763038 573 browser details YourSeq 102 943 1248 3000 82.6% chr6 + 120340094 120340385 292 browser details YourSeq 100 1887 2024 3000 85.3% chr1 - 87361041 87361174 134 browser details YourSeq 95 1887 2201 3000 74.3% chr11 + 89565488 89565639 152 browser details YourSeq 94 945 1099 3000 94.3% chr15 + 26754170 26754325 156 browser details YourSeq 93 1148 1481 3000 89.1% chr13 + 55182882 55183374 493 browser details YourSeq 92 1133 1477 3000 79.7% chr11 - 100617115 100617440 326 browser details YourSeq 92 945 1231 3000 91.9% chr11 - 62498290 62498755 466 browser details YourSeq 91 1456 2007 3000 95.1% chr10 + 92729939 92730859 921 browser details YourSeq 88 1898 2079 3000 83.1% chr2 - 54999435 54999593 159 browser details YourSeq 88 1748 2028 3000 77.4% chr3 + 91800682 91800807 126 browser details YourSeq 85 1897 2024 3000 84.0% chr5 + 122083711 122083832 122 browser details YourSeq 85 1042 1248 3000 93.0% chr5 + 20869268 20869566 299 browser details YourSeq 85 1882 2008 3000 81.1% chr11 + 87714186 87714306 121 browser details YourSeq 84 1900 2035 3000 80.8% chr14 - 32385038 32385163 126 Note: The 3000 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 3000 1 3000 3000 100.0% chr6 + 128406824 128409823 3000 browser details YourSeq 72 294 415 3000 92.1% chr4 + 45955764 45955896 133 browser details YourSeq 68 298 414 3000 90.5% chr14 - 24062999 24063126 128 browser details YourSeq 67 296 423 3000 87.7% chr1 + 59672892 59673037 146 browser details YourSeq 65 303 431 3000 98.6% chr17 + 33636575 33637010 436 browser details YourSeq 63 299 405 3000 92.0% chr18 + 7500657 7500780 124 browser details YourSeq 57 304 412 3000 96.8% chr2 + 105653840 105653972 133 browser details YourSeq 57 289 416 3000 92.7% chr14 + 31723123 31723252 130 browser details YourSeq 55 294 414 3000 89.9% chr2 - 125850014 125850139 126 browser details YourSeq 54 298 405 3000 78.7% chr5 - 72208539 72208629 91 browser details YourSeq 52 288 404 3000 93.6% chrX + 48667826 48667951 126 browser details YourSeq 49 298 396 3000 92.9% chr1 + 10960746 10961056 311 browser details YourSeq 47 294 402 3000 92.8% chr11 - 87732593 87732713 121 browser details YourSeq 47 289 400 3000 72.6% chr10 - 79942735 79942805 71 browser details YourSeq 47 289 404 3000 96.2% chr1 - 34435998 34436113 116 browser details YourSeq 44 290 333 3000 100.0% chr4 - 44050628 44050671 44 browser details YourSeq 44 289 402 3000 90.2% chr11 - 79794851 79794963 113 browser details YourSeq 44 2791 2972 3000 98.0% chr11 - 9332962 9333144 183 browser details YourSeq 43 2345 2516 3000 89.1% chr10 - 69747344 69747809 466 browser details YourSeq 43 289 332 3000 100.0% chr2 + 165977969 165978013 45 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Nrip2 nuclear receptor interacting protein 2 [ Mus musculus (house mouse) ] Gene ID: 60345, updated on 12-Aug-2019 Gene summary Official Symbol Nrip2 provided by MGI Official Full Name nuclear receptor interacting protein 2 provided by MGI Primary source MGI:MGI:1891884 See related Ensembl:ENSMUSG00000001520 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 NIX1; AW491344 Expression Broad expression in cerebellum adult (RPKM 9.0), frontal lobe adult (RPKM 3.6) and 18 other tissues See more Orthologs human all Genomic context Location: 6; 6 F3 See Nrip2 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (128397818..128409780) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (128349784..128358953) Chromosome 6 - NC_000072.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Nrip2 ENSMUSG00000001520 Description nuclear receptor interacting protein 2 [Source:MGI Symbol;Acc:MGI:1891884] Gene Synonyms NIX1 Location Chromosome 6: 128,399,296-128,416,427 forward strand. GRCm38:CM000999.2 About this gene This gene has 6 transcripts (splice variants), 161 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nrip2- ENSMUST00000001561.11 1893 229aa ENSMUSP00000001561.5 Protein coding CCDS20571 Q9JHR9 TSL:1 201 GENCODE basic APPRIS P1 Nrip2- ENSMUST00000120405.3 1468 270aa ENSMUSP00000113317.1 Protein coding CCDS51917 Q9JHR9 TSL:2 202 GENCODE basic Nrip2- ENSMUST00000123867.7 668 219aa ENSMUSP00000122558.1 Protein coding - E9QAD1 CDS 3' 203 incomplete TSL:5 Nrip2- ENSMUST00000204836.1 572 190aa ENSMUSP00000144750.1 Non stop decay - A0A0N4SUN5 CDS 5' 206 incomplete TSL:5 Nrip2- ENSMUST00000147155.7 774 113aa ENSMUSP00000122305.1 Nonsense mediated - F7BIJ5 CDS 5' 205 decay incomplete TSL:5 Nrip2- ENSMUST00000136631.1 3261 No - Retained intron - - TSL:2 204 protein Page 6 of 8 https://www.alphaknockout.com 37.13 kb Forward strand 128.39Mb 128.40Mb 128.41Mb 128.42Mb Genes Gm44596-201 >protein coding (Comprehensive set... Nrip2-203 >protein coding Nrip2-205 >nonsense mediated decay Nrip2-201 >protein coding Nrip2-202 >protein coding Nrip2-204 >retained intron Nrip2-206 >non stop decay Contigs AC116573.7 > Genes < Gm15862-201lncRNA < Itfg2-201protein coding (Comprehensive set... < Itfg2-208protein coding < Itfg2-202retained intron < Itfg2-203protein coding < Itfg2-206protein coding < Itfg2-209retained intron < Itfg2-210retained intron < Itfg2-205retained intron < Itfg2-207nonsense mediated decay < Itfg2-204nonsense mediated decay Regulatory Build 128.39Mb 128.40Mb 128.41Mb 128.42Mb Reverse strand 37.13 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000001561 9.17 kb Forward strand Nrip2-201 >protein coding ENSMUSP00000001... MobiDB lite Low complexity (Seg) Superfamily Aspartic peptidase domain superfamily Pfam Aspartic peptidase, DDI1-type PANTHER PTHR12917:SF17 PTHR12917 Gene3D Aspartic peptidase domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 200 229 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.