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Mouse Usp24 Knockout Project (CRISPR/Cas9)

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Mouse Usp24 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Usp24 Knockout Project (CRISPR/Cas9) Objective: To create a Usp24 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Usp24 gene (NCBI Reference Sequence: NM_183225 ; Ensembl: ENSMUSG00000028514 ) is located on Mouse chromosome 4. 68 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 68 (Transcript: ENSMUST00000094933). Exon 2 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 2 starts from about 4.02% of the coding region. Exon 2 covers 2.11% of the coding region. The size of effective KO region: ~166 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 gRNA region 5' gRNA region 3' 1 2 68 Legends Exon of mouse Usp24 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 962 bp section downstream 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. 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(29.65% 593) | C(17.75% 355) | T(26.1% 522) | G(26.5% 530) 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(962bp) | A(29.63% 285) | C(18.3% 176) | T(31.19% 300) | G(20.89% 201) Note: The 962 bp section downstream 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. 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% chr4 + 106339215 106341214 2000 browser details YourSeq 150 1060 1458 2000 91.6% chr5 - 71231396 71231869 474 browser details YourSeq 149 950 1454 2000 88.4% chr19 - 3688449 3688937 489 browser details YourSeq 139 1307 1462 2000 94.9% chr12 - 78679535 78679691 157 browser details YourSeq 138 1304 1459 2000 93.0% chr7 - 101760493 101760647 155 browser details YourSeq 137 1307 1460 2000 94.8% chr11 - 75457792 75457945 154 browser details YourSeq 136 1263 1454 2000 92.5% chr1 - 33089798 33090028 231 browser details YourSeq 136 1304 1475 2000 92.1% chr18 + 16280401 16280578 178 browser details YourSeq 135 1307 1462 2000 93.6% chrX - 38500549 38500706 158 browser details YourSeq 135 1305 1459 2000 92.3% chr3 - 7446988 7447141 154 browser details YourSeq 135 1304 1464 2000 92.5% chr14 + 46812975 46813138 164 browser details YourSeq 134 1062 1451 2000 80.6% chr8 - 69849845 69850043 199 browser details YourSeq 134 1303 1450 2000 96.0% chr19 + 16886149 16886309 161 browser details YourSeq 134 1303 1459 2000 93.0% chr11 + 5965306 5965481 176 browser details YourSeq 133 1304 1458 2000 93.0% chr11 - 115468885 115469039 155 browser details YourSeq 132 1305 1458 2000 92.9% chr8 - 90474949 90475102 154 browser details YourSeq 132 1312 1460 2000 94.7% chr8 - 44723648 44723803 156 browser details YourSeq 132 1306 1458 2000 93.5% chr12 - 13497774 13497926 153 browser details YourSeq 132 1312 1462 2000 94.1% chr6 + 106797183 106797336 154 browser details YourSeq 132 1304 1460 2000 93.0% chr17 + 30022106 30022262 157 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 962 1 962 962 100.0% chr4 + 106341381 106342342 962 browser details YourSeq 27 876 915 962 89.7% chr7 + 67594730 67594768 39 browser details YourSeq 25 678 711 962 78.2% chr10 + 67379786 67379817 32 browser details YourSeq 24 679 704 962 88.0% chr8 - 31771786 31771810 25 browser details YourSeq 23 590 618 962 89.7% chr13 - 29915615 29915643 29 browser details YourSeq 23 677 705 962 89.7% chr1 - 119103023 119103051 29 browser details YourSeq 22 684 711 962 89.3% chr6 - 77206209 77206236 28 browser details YourSeq 22 678 703 962 87.5% chr11 - 102705941 102705965 25 browser details YourSeq 22 880 901 962 100.0% chr11 - 40897875 40897896 22 browser details YourSeq 22 683 714 962 84.4% chr1 - 186798601 186798632 32 browser details YourSeq 22 691 712 962 100.0% chr7 + 114583550 114583571 22 browser details YourSeq 22 678 703 962 92.4% chr1 + 74181401 74181426 26 browser details YourSeq 21 581 601 962 100.0% chr5 - 3228616 3228636 21 browser details YourSeq 21 273 293 962 100.0% chr1 - 72172137 72172157 21 browser details YourSeq 21 692 712 962 100.0% chr2 + 164648846 164648866 21 browser details YourSeq 21 676 696 962 100.0% chr11 + 70466891 70466911 21 browser details YourSeq 21 683 703 962 100.0% chr1 + 74235118 74235138 21 browser details YourSeq 20 595 614 962 100.0% chr10 - 124055572 124055591 20 browser details YourSeq 20 691 712 962 95.5% chr10 - 118763675 118763696 22 browser details YourSeq 20 583 602 962 100.0% chr10 - 71085226 71085245 20 Note: The 962 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Usp24 ubiquitin specific peptidase 24 [ Mus musculus (house mouse) ] Gene ID: 329908, updated on 10-Oct-2019 Gene summary Official Symbol Usp24 provided by MGI Official Full Name ubiquitin specific peptidase 24 provided by MGI Primary source MGI:MGI:1919936 See related Ensembl:ENSMUSG00000028514 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 C79851; AI414051; B130021E18; 2700066K03Rik; 2810030C21Rik Expression Ubiquitous expression in kidney adult (RPKM 8.3), thymus adult (RPKM 7.3) and 28 other tissues See more Orthologs human all Genomic context Location: 4; 4 C7 See Usp24 in Genome Data Viewer Exon count: 70 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (106315980..106446613) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (105988818..106113932) Chromosome 4 - NC_000070.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Usp24 ENSMUSG00000028514 Description ubiquitin specific peptidase 24 [Source:MGI Symbol;Acc:MGI:1919936] Gene Synonyms 2700066K03Rik, 2810030C21Rik Location Chromosome 4: 106,316,213-106,441,322 forward strand. GRCm38:CM000997.2 About this gene This gene has 4 transcripts (splice variants), 164 orthologues, 49 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Usp24-201 ENSMUST00000094933.4 10575 2617aa ENSMUSP00000092538.4 Protein coding CCDS51251 B1AY13 TSL:5 GENCODE basic APPRIS P2 Usp24-204 ENSMUST00000165709.7 10594 2618aa ENSMUSP00000133095.1 Protein coding - E9PV45 TSL:5 GENCODE basic APPRIS ALT2 Usp24-202 ENSMUST00000106798.6 3179 No protein - Retained intron - - TSL:1 Usp24-203 ENSMUST00000150521.1 389 No protein - lncRNA - - TSL:3 145.11 kb Forward strand 106.32Mb 106.34Mb 106.36Mb 106.38Mb 106.40Mb 106.42Mb 106.44Mb Genes (Comprehensive set... Usp24-204 >protein coding Usp24-202 >retained intron Usp24-203 >lncRNA Usp24-201 >protein coding Contigs AL840623.10 > AL954352.10 > Genes < Pcsk9-201protein coding (Comprehensive set... Regulatory Build 106.32Mb 106.34Mb 106.36Mb 106.38Mb 106.40Mb 106.42Mb 106.44Mb Reverse strand 145.11 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: ENSMUST00000094933 125.09 kb Forward strand Usp24-201 >protein coding ENSMUSP00000092... MobiDB lite Low complexity (Seg) Superfamily UBA-like superfamily Ubiquitin-like domain superfamily Papain-like cysteine peptidase superfamily Armadillo-type fold Pfam Peptidase C19, ubiquitin carboxyl-terminal hydrolase PROSITE profiles Ubiquitin-associated domain Ubiquitin specific protease domain PROSITE patterns Ubiquitin specific protease, conserved site Ubiquitin specific protease, conserved site PANTHER Ubiquitin-specific peptidase 24 PTHR24006 Gene3D 1.10.8.10 3.10.20.90 3.90.70.10 CDD cd14286 cd17065 cd02659 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 400 800 1200 1600 2000 2617 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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