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Mouse Kcnrg Conditional Knockout Project (CRISPR/Cas9)

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Mouse Kcnrg Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Kcnrg Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Kcnrg conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Kcnrg gene (NCBI Reference Sequence: NM_206974 ; Ensembl: ENSMUSG00000046168 ) is located on Mouse chromosome 14. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000225582). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kcnrg gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-460E11 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 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of intron 1 for 3'-loxP site insertion: 3545 bp. The size of effective cKO region: ~833 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele A gRNA region T 5' G 3' 3 1 2 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Exon of mouse Trim13 Homology arm Exon of mouse Kcnrg 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(7073bp) | A(29.24% 2068) | C(19.74% 1396) | T(30.17% 2134) | G(20.85% 1475) 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% chr14 + 61604514 61607513 3000 browser details YourSeq 1316 30 1430 3000 97.2% chr12 + 31418855 31420252 1398 browser details YourSeq 87 796 919 3000 85.6% chr14 + 61603955 61604072 118 browser details YourSeq 23 1411 1438 3000 80.8% chr7 - 31613036 31613061 26 browser details YourSeq 23 1411 1438 3000 80.8% chr7 - 32087466 32087491 26 browser details YourSeq 22 2281 2302 3000 100.0% chr10 - 84710835 84710856 22 browser details YourSeq 21 984 1004 3000 100.0% chr9 - 22847916 22847936 21 Note: The 3000 bp section upstream of Exon 1 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% chr14 + 61608337 61611336 3000 browser details YourSeq 182 673 1548 3000 84.5% chr17 + 88259066 88259724 659 browser details YourSeq 181 673 1212 3000 94.2% chr4 + 137607898 137624244 16347 browser details YourSeq 171 1073 2831 3000 94.8% chrX + 136613222 137086155 472934 browser details YourSeq 150 1075 1552 3000 85.4% chr11 - 79813531 79813701 171 browser details YourSeq 141 1074 1550 3000 84.5% chr2 - 39325104 39325257 154 browser details YourSeq 134 1074 1229 3000 91.8% chr8 + 60694463 60694612 150 browser details YourSeq 131 1058 1210 3000 94.6% chr11 + 115480504 115480657 154 browser details YourSeq 130 2832 2995 3000 90.6% chr2 + 109916033 109916197 165 browser details YourSeq 129 2839 2992 3000 92.2% chr12 - 52713799 52713961 163 browser details YourSeq 128 1084 1229 3000 92.7% chr9 + 118460517 118460656 140 browser details YourSeq 128 1080 1229 3000 93.9% chr6 + 10576268 10576427 160 browser details YourSeq 128 2777 2993 3000 84.9% chr1 + 36584889 36585035 147 browser details YourSeq 127 2777 2995 3000 84.3% chr1 - 135285243 135285391 149 browser details YourSeq 127 2836 2989 3000 89.9% chr7 + 45737903 45738053 151 browser details YourSeq 126 1070 1209 3000 95.0% chr5 - 43697661 43697800 140 browser details YourSeq 126 2777 2995 3000 83.8% chr3 - 44751495 44751651 157 browser details YourSeq 126 1075 1210 3000 96.4% chr13 - 73858233 73858368 136 browser details YourSeq 125 2838 2982 3000 91.7% chr16 + 56170833 56170976 144 browser details YourSeq 124 2775 2992 3000 83.3% chr10 - 77639475 77639635 161 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Kcnrg potassium channel regulator [ Mus musculus (house mouse) ] Gene ID: 328424, updated on 24-Oct-2019 Gene summary Official Symbol Kcnrg provided by MGI Official Full Name potassium channel regulator provided by MGI Primary source MGI:MGI:2685591 See related Ensembl:ENSMUSG00000046168 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 Clld4; Gm745; E030012H22Rik Expression Broad expression in lung adult (RPKM 1.7), limb E14.5 (RPKM 1.3) and 23 other tissues See more Orthologs human all Genomic context Location: 14; 14 D1 See Kcnrg in Genome Data Viewer Exon count: 2 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (61607457..61612833) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (62226294..62231670) Chromosome 14 - NC_000080.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Kcnrg ENSMUSG00000046168 Description potassium channel regulator [Source:MGI Symbol;Acc:MGI:2685591] Gene Synonyms E030012H22Rik, LOC328424 Location Chromosome 14: 61,607,457-61,612,826 forward strand. GRCm38:CM001007.2 About this gene This gene has 2 transcripts (splice variants), 124 orthologues, 14 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Kcnrg-201 ENSMUST00000051184.9 1766 264aa ENSMUSP00000055327.8 Protein coding CCDS27187 Q2TUM3 TSL:1 GENCODE basic APPRIS P2 Kcnrg-202 ENSMUST00000225582.1 1821 191aa ENSMUSP00000153429.1 Protein coding - Q2TUM3 GENCODE basic APPRIS ALT2 25.37 kb Forward strand 61.60Mb 61.61Mb 61.62Mb Genes Trim13-202 >protein coding Kcnrg-202 >protein coding (Comprehensive set... Trim13-201 >protein coding Kcnrg-201 >protein coding Contigs AC154660.2 > Genes < Dleu2-208lncRNA (Comprehensive set... < Dleu2-203lncRNA < Dleu2-211lncRNA < Dleu2-210lncRNA < Dleu2-205lncRNA < Dleu2-214TEC < Dleu2-201lncRNA Regulatory Build 61.60Mb 61.61Mb 61.62Mb Reverse strand 25.37 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000225582 5.37 kb Forward strand Kcnrg-202 >protein coding ENSMUSP00000153... Superfamily SKP1/BTB/POZ domain superfamily SMART BTB/POZ domain Pfam Potassium channel tetramerisation-type BTB domain PANTHER PTHR14499 PTHR14499:SF5 Gene3D 3.30.710.10 CDD cd18375 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 191 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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