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Mouse Nol4 Knockout Project (CRISPR/Cas9)

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Mouse Nol4 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Nol4 Knockout Project (CRISPR/Cas9) Objective: To create a Nol4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nol4 gene (NCBI Reference Sequence: NM_199024 ; Ensembl: ENSMUSG00000041923 ) is located on Mouse chromosome 18. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000097651). Exon 3~5 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: Exon 3 starts from about 28.64% of the coding region. Exon 3~5 covers 24.71% of the coding region. The size of effective KO region: ~9376 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 3 4 5 10 Legends Exon of mouse Nol4 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 3 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 5 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(27.15% 543) | C(19.65% 393) | T(34.2% 684) | G(19.0% 380) Note: The 2000 bp section upstream of Exon 3 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(30.0% 600) | C(18.4% 368) | T(35.7% 714) | G(15.9% 318) Note: The 2000 bp section downstream of Exon 5 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% chr18 - 22921958 22923957 2000 browser details YourSeq 179 74 744 2000 77.5% chr3 - 66640535 66641170 636 browser details YourSeq 155 23 748 2000 90.3% chr12 - 72319871 72403459 83589 browser details YourSeq 147 328 778 2000 78.9% chr14 - 62623597 62624081 485 browser details YourSeq 147 61 674 2000 76.9% chr3 + 90563295 90563872 578 browser details YourSeq 143 331 748 2000 82.0% chr15 - 57850522 57850976 455 browser details YourSeq 141 366 778 2000 82.0% chr18 - 33304601 33305034 434 browser details YourSeq 138 71 770 2000 89.3% chr9 + 84013763 84014788 1026 browser details YourSeq 135 74 778 2000 79.2% chrX + 100128097 100128818 722 browser details YourSeq 123 554 776 2000 87.3% chrX + 85425626 85425856 231 browser details YourSeq 121 400 778 2000 79.4% chr1 + 7248174 7248607 434 browser details YourSeq 114 101 748 2000 79.8% chr11 + 72694998 72695630 633 browser details YourSeq 113 95 679 2000 71.7% chrX + 18849050 18849322 273 browser details YourSeq 110 109 778 2000 79.0% chr13 + 94031809 94032452 644 browser details YourSeq 110 518 778 2000 79.4% chr1 + 23452400 23452651 252 browser details YourSeq 109 378 683 2000 76.9% chr19 - 38083405 38083772 368 browser details YourSeq 107 372 777 2000 81.0% chr3 - 41865350 41865807 458 browser details YourSeq 105 554 749 2000 81.6% chr8 - 27220625 27220837 213 browser details YourSeq 104 100 778 2000 78.2% chr1 - 61501657 61502312 656 browser details YourSeq 102 554 748 2000 80.4% chr1 - 6579436 6579637 202 Note: The 2000 bp section upstream of Exon 3 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% chr18 - 22910582 22912581 2000 browser details YourSeq 23 415 443 2000 89.7% chr4 + 79646460 79646488 29 browser details YourSeq 22 1376 1397 2000 100.0% chr1 + 161961065 161961086 22 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Nol4 nucleolar protein 4 [ Mus musculus (house mouse) ] Gene ID: 319211, updated on 12-Aug-2019 Gene summary Official Symbol Nol4 provided by MGI Official Full Name nucleolar protein 4 provided by MGI Primary source MGI:MGI:2441684 See related Ensembl:ENSMUSG00000041923 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 Gm1262; 1700013J13Rik; 4930568N03Rik Expression Biased expression in frontal lobe adult (RPKM 10.4), CNS E18 (RPKM 10.4) and 6 other tissues See more Orthologs human all Genomic context Location: 18; 18 A2 See Nol4 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (22693152..23042640, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (22851656..23200154, complement) Chromosome 18 - NC_000084.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Nol4 ENSMUSG00000041923 Description nucleolar protein 4 [Source:MGI Symbol;Acc:MGI:2441684] Gene Synonyms 1700013J13Rik, 4930568N03Rik, LOC383304 Location Chromosome 18: 22,693,181-23,041,653 reverse strand. GRCm38:CM001011.2 About this gene This gene has 9 transcripts (splice variants), 143 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nol4-204 ENSMUST00000097651.9 4549 483aa ENSMUSP00000095256.3 Protein coding CCDS29093 P60954 TSL:1 GENCODE basic Nol4-202 ENSMUST00000081423.12 3309 564aa ENSMUSP00000080150.6 Protein coding CCDS50235 P60954 TSL:1 GENCODE basic Nol4-206 ENSMUST00000164186.7 2622 637aa ENSMUSP00000130950.1 Protein coding CCDS84363 E9Q947 TSL:5 GENCODE basic APPRIS P4 Nol4-208 ENSMUST00000164893.7 2175 573aa ENSMUSP00000127870.1 Protein coding CCDS84362 G3UW35 TSL:5 GENCODE basic APPRIS ALT1 Nol4-203 ENSMUST00000092015.10 2827 355aa ENSMUSP00000089642.4 Protein coding - F6XSA1 CDS 5' incomplete TSL:1 Nol4-201 ENSMUST00000069215.12 1902 419aa ENSMUSP00000064166.6 Protein coding - F7BIN0 CDS 5' incomplete TSL:1 Nol4-209 ENSMUST00000165323.1 477 108aa ENSMUSP00000125860.1 Protein coding - E9Q5X0 CDS 3' incomplete TSL:5 Nol4-207 ENSMUST00000164521.1 345 No protein - lncRNA - - TSL:2 Nol4-205 ENSMUST00000097652.3 277 No protein - lncRNA - - TSL:5 Page 7 of 9 https://www.alphaknockout.com 368.47 kb Forward strand 22.7Mb 22.8Mb 22.9Mb 23.0Mb Contigs AC103368.7 > AC131338.4 > Genes (Comprehensive set... < Nol4-203protein coding < Nol4-209protein coding < Nol4-202protein coding < Nol4-204protein coding < Nol4-201protein coding < Nol4-205lncRNA < Nol4-208protein coding < Nol4-206protein coding < Nol4-207lncRNA Regulatory Build 22.7Mb 22.8Mb 22.9Mb 23.0Mb Reverse strand 368.47 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000097651 < Nol4-204protein coding Reverse strand 348.02 kb ENSMUSP00000095... MobiDB lite Low complexity (Seg) PANTHER Nucleolar protein 4 family Nucleolar protein 4 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 483 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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