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Mouse Nol4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Nol4 conditional 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 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nol4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-318A5 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 3 starts from about 28.64% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 30203 bp, and the size of intron 3 for 3'-loxP site insertion: 918 bp. The size of effective cKO region: ~612 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 3 4 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Nol4 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. 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(7112bp) | A(30.16% 2145) | C(17.74% 1262) | T(33.9% 2411) | G(18.19% 1294)

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% chr18 - 22922208 22925207 3000 browser details YourSeq 179 1324 1994 3000 77.5% chr3 - 66640535 66641170 636 browser details YourSeq 155 1273 1998 3000 90.3% chr12 - 72319871 72403459 83589 browser details YourSeq 147 1578 2028 3000 78.9% chr14 - 62623597 62624081 485 browser details YourSeq 147 1311 1924 3000 76.9% chr3 + 90563295 90563872 578 browser details YourSeq 143 1581 1998 3000 82.0% chr15 - 57850522 57850976 455 browser details YourSeq 141 1616 2028 3000 82.0% chr18 - 33304601 33305034 434 browser details YourSeq 138 1321 2020 3000 89.3% chr9 + 84013763 84014788 1026 browser details YourSeq 135 1324 2028 3000 79.2% chrX + 100128097 100128818 722 browser details YourSeq 123 1804 2026 3000 87.3% chrX + 85425626 85425856 231 browser details YourSeq 121 1650 2028 3000 79.4% chr1 + 7248174 7248607 434 browser details YourSeq 114 1351 1998 3000 79.8% chr11 + 72694998 72695630 633 browser details YourSeq 113 1345 1929 3000 71.7% chrX + 18849050 18849322 273 browser details YourSeq 110 1359 2028 3000 79.0% chr13 + 94031809 94032452 644 browser details YourSeq 110 1768 2028 3000 79.4% chr1 + 23452400 23452651 252 browser details YourSeq 109 1628 1933 3000 76.9% chr19 - 38083405 38083772 368 browser details YourSeq 107 1622 2027 3000 81.0% chr3 - 41865350 41865807 458 browser details YourSeq 105 1804 1999 3000 81.6% chr8 - 27220625 27220837 213 browser details YourSeq 104 1350 2028 3000 78.2% chr1 - 61501657 61502312 656 browser details YourSeq 102 1804 1998 3000 80.4% chr1 - 6579436 6579637 202

Note: The 3000 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 3000 1 3000 3000 100.0% chr18 - 22918596 22921595 3000 browser details YourSeq 106 1363 1579 3000 93.6% chr5 - 117501424 117501778 355 browser details YourSeq 106 1363 1578 3000 94.2% chr14 + 10859809 10860075 267 browser details YourSeq 100 1364 1582 3000 94.9% chr2 - 172275123 172275612 490 browser details YourSeq 99 1362 1507 3000 93.9% chr9 + 98308870 98309605 736 browser details YourSeq 93 1362 1535 3000 92.1% chr18 - 77196271 77196675 405 browser details YourSeq 88 1369 1502 3000 94.1% chr10 + 83089361 83089560 200 browser details YourSeq 86 1363 1539 3000 90.9% chr16 - 96778208 96778593 386 browser details YourSeq 86 1368 1539 3000 93.3% chr13 + 46162731 46163221 491 browser details YourSeq 81 1362 1578 3000 79.6% chr3 - 104690106 104690201 96 browser details YourSeq 79 1376 1583 3000 87.7% chr5 - 17655079 17655280 202 browser details YourSeq 78 1362 1538 3000 96.6% chr1 - 88973094 88973600 507 browser details YourSeq 78 1363 1590 3000 77.8% chr1 + 56265751 56265861 111 browser details YourSeq 73 1386 1523 3000 79.4% chr18 + 78746803 78746906 104 browser details YourSeq 73 1363 1492 3000 94.2% chr11 + 43795541 43795675 135 browser details YourSeq 72 1366 1579 3000 82.1% chr4 + 118510973 118511097 125 browser details YourSeq 72 1378 1574 3000 95.1% chr1 + 40674925 40675147 223 browser details YourSeq 71 1363 1495 3000 80.3% chr4 - 8631879 8631971 93 browser details YourSeq 68 1363 1529 3000 97.3% chr13 - 20250642 20250841 200 browser details YourSeq 67 1369 1539 3000 78.1% chr13 - 49153330 49153435 106

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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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

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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

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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

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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.

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