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Mouse Cnih1 Knockout Project (CRISPR/Cas9)

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Mouse Cnih1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Cnih1 Knockout Project (CRISPR/Cas9) Objective: To create a Cnih1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Cnih1 gene (NCBI Reference Sequence: NM_009919 ; Ensembl: ENSMUSG00000015759 ) is located on Mouse chromosome 14. 5 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 5 (Transcript: ENSMUST00000015903). Exon 2~4 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: The male homozygous mutant mice exhibited a decreased anxiety-like response during open field testing when compared with controls. The male mutants also exhibited decreased body weight, total tissue mass, and lean body mass. Exon 2 starts from about 18.98% of the coding region. Exon 2~4 covers 75.46% of the coding region. The size of effective KO region: ~5292 bp. The KO 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 Legends Exon of mouse Cnih1 Knockout region Page 2 of 8 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. 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 4 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.95% 539) | C(18.65% 373) | T(32.3% 646) | G(22.1% 442) 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(24.85% 497) | C(19.95% 399) | T(31.95% 639) | G(23.25% 465) Note: The 2000 bp section downstream of Exon 4 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 8 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% chr14 - 46784181 46786180 2000 browser details YourSeq 45 520 583 2000 80.8% chr14 - 114407163 114407217 55 browser details YourSeq 43 539 582 2000 100.0% chr17 + 63844770 63844814 45 browser details YourSeq 42 539 583 2000 97.8% chr6 - 122900861 122900906 46 browser details YourSeq 41 539 582 2000 97.8% chr7 - 95940640 95940684 45 browser details YourSeq 41 539 583 2000 97.8% chr15 - 89975023 89975071 49 browser details YourSeq 41 539 584 2000 95.7% chr15 - 26221429 26221475 47 browser details YourSeq 41 539 582 2000 97.8% chr14 - 111939584 111939628 45 browser details YourSeq 41 539 582 2000 97.8% chr11 - 54419948 54419992 45 browser details YourSeq 41 539 582 2000 97.7% chr10 - 106182534 106182578 45 browser details YourSeq 41 539 582 2000 97.8% chr1 - 143070835 143070879 45 browser details YourSeq 41 539 582 2000 97.8% chr9 + 101245940 101245984 45 browser details YourSeq 41 539 582 2000 97.8% chr16 + 47047599 47047643 45 browser details YourSeq 41 539 582 2000 97.7% chr14 + 82627947 82627991 45 browser details YourSeq 41 539 582 2000 97.8% chr12 + 103210919 103210963 45 browser details YourSeq 41 539 582 2000 97.8% chr12 + 43605965 43606009 45 browser details YourSeq 40 539 582 2000 97.8% chr8 - 103307202 103307251 50 browser details YourSeq 40 539 583 2000 97.8% chr8 - 13829268 13829321 54 browser details YourSeq 40 539 579 2000 100.0% chr7 - 36655757 36655798 42 browser details YourSeq 40 541 581 2000 100.0% chr16 - 74832616 74832657 42 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% chr14 - 46776889 46778888 2000 browser details YourSeq 75 1003 1130 2000 93.3% chr5 - 24068521 24068653 133 browser details YourSeq 65 920 1144 2000 90.3% chr4 + 153622132 153622363 232 browser details YourSeq 63 1003 1127 2000 92.2% chr6 - 145134972 145135119 148 browser details YourSeq 58 1037 1125 2000 79.4% chr6 + 88217722 88217808 87 browser details YourSeq 57 1071 1142 2000 91.0% chr5 - 150995193 150995263 71 browser details YourSeq 56 1063 1133 2000 91.4% chr19 + 10568673 10568755 83 browser details YourSeq 54 1016 1106 2000 78.6% chr4 - 149615013 149615101 89 browser details YourSeq 54 1045 1133 2000 93.7% chr10 - 75191259 75191359 101 browser details YourSeq 54 1045 1125 2000 89.9% chr11 + 60307758 60307838 81 browser details YourSeq 53 1016 1120 2000 91.0% chr6 + 121620842 121620949 108 browser details YourSeq 52 1014 1127 2000 80.6% chr4 - 8743389 8743495 107 browser details YourSeq 52 1003 1142 2000 70.3% chr9 + 63744236 63744377 142 browser details YourSeq 52 1068 1134 2000 89.3% chr4 + 12056954 12057030 77 browser details YourSeq 52 1069 1142 2000 81.3% chr2 + 23122175 23122244 70 browser details YourSeq 51 1004 1103 2000 87.1% chr7 - 16607428 16607526 99 browser details YourSeq 51 1047 1126 2000 82.5% chr14 - 31045923 31046004 82 browser details YourSeq 51 1040 1119 2000 84.0% chr7 + 19689310 19689391 82 browser details YourSeq 51 1016 1121 2000 88.3% chr12 + 86732280 86732397 118 browser details YourSeq 51 1045 1125 2000 83.1% chr10 + 89391512 89391591 80 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Cnih1 cornichon family AMPA receptor auxiliary protein 1 [ Mus musculus (house mouse) ] Gene ID: 12793, updated on 24-Oct-2019 Gene summary Official Symbol Cnih1 provided by MGI Official Full Name cornichon family AMPA receptor auxiliary protein 1 provided by MGI Primary source MGI:MGI:1277202 See related Ensembl:ENSMUSG00000015759 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 Cnih; CNIH-1; 0610007J15 Expression Ubiquitous expression in adrenal adult (RPKM 63.7), placenta adult (RPKM 36.7) and 28 other tissues See more Orthologs human all Genomic context Location: 14; 14 C1 See Cnih1 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (46775567..46788357, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (47395242..47408032, complement) Chromosome 14 - NC_000080.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Cnih1 ENSMUSG00000015759 Description cornichon family AMPA receptor auxiliary protein 1 [Source:MGI Symbol;Acc:MGI:1277202] Gene Synonyms Cnih Location Chromosome 14: 46,775,567-46,788,411 reverse strand. GRCm38:CM001007.2 About this gene This gene has 3 transcripts (splice variants), 222 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Cnih1-201 ENSMUST00000015903.11 1538 144aa ENSMUSP00000015903.4 Protein coding CCDS26980 O35372 TSL:1 GENCODE basic APPRIS P1 Cnih1-202 ENSMUST00000146629.2 789 160aa ENSMUSP00000116885.2 Protein coding - D6RGU4 TSL:5 GENCODE basic Cnih1-203 ENSMUST00000227583.1 781 No protein - Retained intron - - - 32.84 kb Forward strand 46.77Mb 46.78Mb 46.79Mb Genes Cdkn3-201 >protein coding Gm48933-201 >lncRNA (Comprehensive set... Cdkn3-203 >protein coding Cdkn3-204 >nonsense mediated decay Cdkn3-202 >lncRNA Contigs < AC093043.6 Genes (Comprehensive set... < Cnih1-201protein coding < Cnih1-203retained intron < Cnih1-202protein coding Regulatory Build 46.77Mb 46.78Mb 46.79Mb Reverse strand 32.84 kb Regulation Legend CTCF 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: ENSMUST00000015903 < Cnih1-201protein coding Reverse strand 12.85 kb ENSMUSP00000015... Transmembrane heli... Low complexity (Seg) SMART Cornichon Pfam Cornichon PROSITE patterns Cornichon, conserved site PANTHER PTHR12290 PTHR12290:SF10 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 20 40 60 80 100 120 144 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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