https://www.alphaknockout.com Mouse Rint1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Rint1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rint1 gene (NCBI Reference Sequence: NM_177323 ; Ensembl: ENSMUSG00000028999 ) is located on Mouse chromosome 5. 15 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000030852). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rint1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-14F17 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: Mice homozygous for a mutant allele exhibit early embryonic lethality. Mice heterozygous for a mutant allele exhibit premature death with a life span of 24 months and increased multiple tumor incidence. Exon 4 starts from about 11.53% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 6107 bp, and the size of intron 4 for 3'-loxP site insertion: 4473 bp. The size of effective cKO region: ~742 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 gRNA region 5' gRNA region 3' 1 4 15 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Rint1 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(7242bp) | A(27.09% 1962) | C(20.33% 1472) | T(30.93% 2240) | G(21.65% 1568) 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% chr5 + 23797346 23800345 3000 browser details YourSeq 836 2137 3000 3000 98.5% chr5 - 15570625 15571494 870 browser details YourSeq 828 2138 3000 3000 98.1% chr4 - 101227883 101228749 867 browser details YourSeq 827 2137 3000 3000 98.2% chr6 - 13090595 13091462 868 browser details YourSeq 821 2137 3000 3000 97.7% chr10 - 89717955 89718826 872 browser details YourSeq 815 2137 3000 3000 98.1% chr6 - 29340348 29341218 871 browser details YourSeq 812 2137 3000 3000 97.5% chrX - 155797527 155798393 867 browser details YourSeq 812 2137 3000 3000 98.0% chr3 + 93952506 93953369 864 browser details YourSeq 809 2140 3000 3000 98.0% chr3 + 94063842 94064702 861 browser details YourSeq 805 2137 3000 3000 96.9% chr8 + 93364793 93365660 868 browser details YourSeq 803 2137 3000 3000 96.8% chr6 + 56823521 56824388 868 browser details YourSeq 803 2139 3000 3000 96.8% chr2 + 26698086 26698954 869 browser details YourSeq 801 2137 3000 3000 96.7% chr7 - 18742599 18743466 868 browser details YourSeq 794 2137 3000 3000 96.3% chr7 + 47497185 47925945 428761 browser details YourSeq 794 1981 3000 3000 94.3% chr6 + 37776851 37777850 1000 browser details YourSeq 793 2139 3000 3000 96.4% chr10 + 26744678 26745542 865 browser details YourSeq 791 2149 3000 3000 96.6% chr2 + 26704894 26705752 859 browser details YourSeq 788 2139 3000 3000 97.5% chr4 + 143084578 143085439 862 browser details YourSeq 785 2137 3000 3000 96.9% chr1 + 7558155 7559020 866 browser details YourSeq 779 2139 3000 3000 97.5% chr15 + 47369084 47369944 861 Note: The 3000 bp section upstream of Exon 4 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% chr5 + 23801088 23804087 3000 browser details YourSeq 204 1615 1839 3000 94.4% chr9 - 57717497 57717712 216 browser details YourSeq 199 1628 1839 3000 97.1% chr17 + 29592532 29592738 207 browser details YourSeq 197 1615 1840 3000 98.6% chr11 - 106126948 106127391 444 browser details YourSeq 196 1644 1839 3000 100.0% chrX - 103587718 103587913 196 browser details YourSeq 196 1634 1839 3000 97.6% chr13 - 98898275 98898480 206 browser details YourSeq 195 1641 1839 3000 98.0% chr15 + 50550265 50550462 198 browser details YourSeq 195 1645 1839 3000 100.0% chr1 + 86402078 86402272 195 browser details YourSeq 194 1646 1839 3000 100.0% chr11 - 96360070 96360263 194 browser details YourSeq 194 1643 1840 3000 98.0% chr10 - 21618198 21618394 197 browser details YourSeq 194 1631 1839 3000 97.0% chr7 + 127508594 127508800 207 browser details YourSeq 194 1642 1837 3000 98.5% chr6 + 149008361 149008555 195 browser details YourSeq 193 1647 1839 3000 100.0% chr8 - 84820357 84820549 193 browser details YourSeq 193 1647 1839 3000 100.0% chr7 - 65478864 65479056 193 browser details YourSeq 193 1647 1839 3000 100.0% chr5 - 93105295 93105487 193 browser details YourSeq 193 1647 1839 3000 100.0% chr4 - 149169859 149170051 193 browser details YourSeq 193 1647 1839 3000 100.0% chr4 - 134939273 134939465 193 browser details YourSeq 193 1647 1839 3000 100.0% chr19 - 12814936 12815128 193 browser details YourSeq 193 1647 1839 3000 100.0% chr15 - 98783742 98783934 193 browser details YourSeq 193 1647 1839 3000 100.0% chr7 + 4300981 4301173 193 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Rint1 RAD50 interactor 1 [ Mus musculus (house mouse) ] Gene ID: 72772, updated on 12-Aug-2019 Gene summary Official Symbol Rint1 provided by MGI Official Full Name RAD50 interactor 1 provided by MGI Primary source MGI:MGI:1916233 See related Ensembl:ENSMUSG00000028999 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 Rint-1; 1500019C06Rik; 2810450M21Rik Expression Ubiquitous expression in placenta adult (RPKM 6.8), limb E14.5 (RPKM 6.4) and 28 other tissues See more Orthologs human all Genomic context Location: 5; 5 A3 See Rint1 in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (23787698..23823584) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (23293562..23326187) Chromosome 5 - NC_000071.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Rint1 ENSMUSG00000028999 Description RAD50 interactor 1 [Source:MGI Symbol;Acc:MGI:1916233] Gene Synonyms 1500019C06Rik, 2810450M21Rik Location Chromosome 5: 23,787,711-23,820,369 forward strand. GRCm38:CM000998.2 About this gene This gene has 9 transcripts (splice variants), 202 orthologues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rint1-201 ENSMUST00000030852.12 3298 792aa ENSMUSP00000030852.6 Protein coding CCDS39025 Q8BZ36 TSL:1 GENCODE basic APPRIS P3 Rint1-203 ENSMUST00000115113.2 3048 734aa ENSMUSP00000110766.2 Protein coding CCDS80220 Q8BZ36 TSL:1 GENCODE basic APPRIS ALT2 Rint1-205 ENSMUST00000120869.5 1886 36aa ENSMUSP00000112671.1 Protein coding - D3YU54 TSL:1 GENCODE basic Rint1-204 ENSMUST00000117783.7 560 60aa ENSMUSP00000112763.1 Protein coding - D3YU07 TSL:1 GENCODE basic Rint1-206 ENSMUST00000124680.7 3363 No protein - Retained intron - - TSL:5 Rint1-209 ENSMUST00000196607.1 2308 No protein - Retained intron - - TSL:NA Rint1-207 ENSMUST00000132260.1 671 No protein - Retained intron - - TSL:3 Rint1-202 ENSMUST00000112256.2 1761 No protein - lncRNA - - TSL:1 Rint1-208 ENSMUST00000144709.1 151 No protein - lncRNA - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 52.66 kb Forward strand 23.78Mb 23.79Mb 23.80Mb 23.81Mb 23.82Mb 23.83Mb Genes Rint1-201 >protein coding (Comprehensive set... Rint1-205 >protein coding Rint1-207 >retained intron Rint1-204 >protein coding Rint1-208 >lncRNA Rint1-203 >protein coding Rint1-206 >retained intron Rint1-209 >retained intron Rint1-202 >lncRNA Gm43054-201 >TEC Contigs AC117614.14 > < AC117663.13 Genes < Pus7-201protein coding < 4933427G23Rik-201lncRNA (Comprehensive set... < Pus7-206protein coding < Pus7-204protein coding < Pus7-208nonsense mediated decay Regulatory Build 23.78Mb 23.79Mb 23.80Mb 23.81Mb 23.82Mb 23.83Mb Reverse strand 52.66 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000030852 32.66 kb Forward strand Rint1-201 >protein coding ENSMUSP00000030... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam RINT-1/Tip20 PROSITE profiles RINT-1/Tip20 PANTHER RINT-1/Tip20 PTHR13520:SF0 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 792 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.