https://www.alphaknockout.com

Mouse Nop58 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Nop58 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nop58 gene (NCBI Reference Sequence: NM_018868 ; Ensembl: ENSMUSG00000026020 ) is located on Mouse chromosome 1. 15 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000191142). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nop58 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-340L12 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 2 starts from about 2.86% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 5259 bp, and the size of intron 2 for 3'-loxP site insertion: 1774 bp. The size of effective cKO region: ~577 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 3 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Nop58 Homology arm cKO region loxP site

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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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7077bp) | A(29.94% 2119) | C(17.1% 1210) | T(33.35% 2360) | G(19.61% 1388)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr1 + 59687237 59690236 3000 browser details YourSeq 234 2206 2538 3000 89.2% chr15 + 8467081 8467545 465 browser details YourSeq 223 2168 2494 3000 91.2% chr1 + 74674272 74674781 510 browser details YourSeq 209 2206 2538 3000 89.7% chr18 + 34227652 34228154 503 browser details YourSeq 205 2164 2888 3000 84.2% chr12 - 3304413 3304912 500 browser details YourSeq 196 2206 2537 3000 88.8% chr11 + 79311697 79312040 344 browser details YourSeq 190 2198 2538 3000 89.6% chr11 - 97595122 97595730 609 browser details YourSeq 189 2206 2494 3000 89.0% chr5 + 101983641 101984162 522 browser details YourSeq 185 2175 2533 3000 91.5% chr17 - 35735337 35735933 597 browser details YourSeq 172 2312 2537 3000 91.0% chr16 + 35794836 35795380 545 browser details YourSeq 169 2241 2536 3000 92.9% chr9 + 65251078 65251664 587 browser details YourSeq 167 2315 2538 3000 89.6% chr8 - 94805463 94805689 227 browser details YourSeq 167 2317 2533 3000 90.0% chr10 - 59996776 59996996 221 browser details YourSeq 166 2311 2539 3000 87.9% chr1 - 134452667 134452890 224 browser details YourSeq 164 2295 2531 3000 89.1% chr15 - 100327002 100327433 432 browser details YourSeq 163 2322 2537 3000 89.1% chr8 + 22742384 22742604 221 browser details YourSeq 162 2312 2527 3000 91.0% chr14 - 65060842 65061081 240 browser details YourSeq 162 2317 2533 3000 88.6% chr14 - 59865045 59865265 221 browser details YourSeq 162 2322 2533 3000 91.4% chr11 - 101207526 101207741 216 browser details YourSeq 162 2294 2527 3000 90.5% chr6 + 120587304 120587633 330

Note: The 3000 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 3000 1 3000 3000 100.0% chr1 + 59690814 59693813 3000 browser details YourSeq 152 1701 2570 3000 93.7% chr1 + 59691939 59745594 53656 browser details YourSeq 124 2345 2571 3000 92.5% chr8 - 90994290 90994576 287 browser details YourSeq 115 2446 2571 3000 96.1% chr5 - 86834080 86834219 140 browser details YourSeq 114 2182 2571 3000 80.5% chr4 - 46026390 46026571 182 browser details YourSeq 113 2168 2571 3000 80.2% chr7 - 53474190 53474325 136 browser details YourSeq 112 2168 2571 3000 82.0% chr3 - 129690313 129690454 142 browser details YourSeq 111 2455 2632 3000 93.0% chr16 + 62056241 62056753 513 browser details YourSeq 110 2446 2571 3000 89.9% chr1 + 165311331 165311448 118 browser details YourSeq 109 2167 2571 3000 79.4% chr18 - 50107790 50107915 126 browser details YourSeq 109 2446 2571 3000 94.4% chr13 + 58632515 58632642 128 browser details YourSeq 108 2446 2571 3000 90.0% chr8 - 70050445 70050563 119 browser details YourSeq 108 2446 2571 3000 93.4% chr7 - 28531643 28531767 125 browser details YourSeq 108 2446 2571 3000 90.0% chr2 - 70671766 70671884 119 browser details YourSeq 107 2168 2571 3000 79.9% chrX - 103988301 103988437 137 browser details YourSeq 107 2446 2571 3000 89.2% chr4 - 115295095 115295214 120 browser details YourSeq 107 2446 2571 3000 93.5% chr4 - 48590694 48590821 128 browser details YourSeq 107 2446 2571 3000 93.5% chr2 - 103619201 103619328 128 browser details YourSeq 107 2446 2571 3000 93.5% chr2 - 26125049 26125176 128 browser details YourSeq 107 2446 2571 3000 93.5% chr14 - 34434945 34435072 128

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Nop58 NOP58 ribonucleoprotein [ Mus musculus (house mouse) ] Gene ID: 55989, updated on 10-Oct-2019

Gene summary

Official Symbol Nop58 provided by MGI Official Full Name NOP58 ribonucleoprotein provided by MGI Primary source MGI:MGI:1933184 See related Ensembl:ENSMUSG00000026020 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 SIK; MSSP; Nol5; nop5 Expression Broad expression in CNS E11.5 (RPKM 41.5), liver E14 (RPKM 35.5) and 20 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 C2 See Nop58 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (59684961..59711510)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (59741850..59768354)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 15 transcripts

Gene: Nop58 ENSMUSG00000026020

Description NOP58 ribonucleoprotein [Source:MGI Symbol;Acc:MGI:1933184] Gene Synonyms MSSP, Nol5, SIK similar protein Location Chromosome 1: 59,684,971-59,719,044 forward strand. GRCm38:CM000994.2 About this gene This gene has 15 transcripts (splice variants), 198 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nop58- ENSMUST00000191142.6 9507 536aa ENSMUSP00000140250.1 Protein coding CCDS35587 Q6DFW4 TSL:1 215 GENCODE basic

Nop58- ENSMUST00000027174.9 1713 444aa ENSMUSP00000027174.4 Protein coding - A0A0A0MQ76 TSL:5 201 GENCODE basic APPRIS P1

Nop58- ENSMUST00000190231.6 639 28aa ENSMUSP00000140581.1 Protein coding - A0A087WRD9 CDS 3' 211 incomplete TSL:2

Nop58- ENSMUST00000187837.6 638 213aa ENSMUSP00000140038.1 Protein coding - A0A087WQ46 CDS 5' and 3' 206 incomplete TSL:5

Nop58- ENSMUST00000188390.1 585 195aa ENSMUSP00000139568.1 Protein coding - A0A087WP00 CDS 5' and 3' 207 incomplete TSL:5

Nop58- ENSMUST00000185772.6 461 20aa ENSMUSP00000139474.1 Protein coding - A0A087WNS6 CDS 3' 203 incomplete TSL:3

Nop58- ENSMUST00000190265.6 714 191aa ENSMUSP00000141100.1 Nonsense mediated - A0A087WSL8 CDS 5' 212 decay incomplete TSL:5

Nop58- ENSMUST00000189327.6 702 60aa ENSMUSP00000139517.1 Nonsense mediated - A0A087WNW0 TSL:5 209 decay

Nop58- ENSMUST00000189919.6 598 146aa ENSMUSP00000141192.1 Nonsense mediated - A0A087WSU5 CDS 5' 210 decay incomplete TSL:3

Nop58- ENSMUST00000187491.1 373 60aa ENSMUSP00000140053.1 Nonsense mediated - A0A087WQ59 CDS 5' 205 decay incomplete TSL:5

Nop58- ENSMUST00000185368.1 891 No - Retained intron - - TSL:3 202 protein

Nop58- ENSMUST00000186044.1 725 No - Retained intron - - TSL:2 204 protein

Nop58- ENSMUST00000189289.6 653 No - Retained intron - - TSL:2 208 protein

Nop58- ENSMUST00000190759.6 623 No - Retained intron - - TSL:3 213 protein

Nop58- ENSMUST00000191088.6 576 No - lncRNA - - TSL:3 214 protein

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54.07 kb Forward strand 59.68Mb 59.69Mb 59.70Mb 59.71Mb 59.72Mb Genes (Comprehensive set... Nop58-214 >lncRNA Nop58-210 >nonsense mediated decay

Nop58-201 >protein coding

Nop58-211 >protein coding Nop58-212 >nonsense mediated decay

Nop58-208 >retained intron Nop58-205 >nonsense mediated decay

Nop58-204 >retained intron Nop58-206 >protein coding

Nop58-215 >protein coding

Nop58-203 >protein coding Gm26287-201 >snoRNA

Nop58-209 >nonsense mediated decay Nop58-207 >protein coding

Nop58-213 >retained intron Snord11-201 >snoRNA

Snord70-201 >snoRNA

Nop58-202 >retained intron

Gm26293-201 >snoRNA

Contigs < AC147254.4 Regulatory Build

59.68Mb 59.69Mb 59.70Mb 59.71Mb 59.72Mb Reverse strand 54.07 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 processed transcript

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Transcript: ENSMUST00000191142

34.04 kb Forward strand

Nop58-215 >protein coding

protein_pic

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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