https://www.alphaknockout.com

Mouse Nprl3 Knockout Project (CRISPR/Cas9)

Objective: To create a Nprl3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nprl3 gene (NCBI Reference Sequence: NM_181569 ; Ensembl: ENSMUSG00000020289 ) is located on Mouse chromosome 11. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000020530). 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: This gene is deleted in the Hbath-J mutation.

Exon 2 starts from about 6.97% of the coding region. Exon 2~4 covers 16.11% of the coding region. The size of effective KO region: ~9220 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 13

Legends Exon of mouse Nprl3 Knockout region

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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. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(23.85% 477) | C(21.7% 434) | T(34.55% 691) | G(19.9% 398)

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(26.55% 531) | C(21.55% 431) | T(30.35% 607) | G(21.55% 431)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr11 - 32263095 32265094 2000 browser details YourSeq 90 115 1116 2000 93.4% chr10 + 80460116 80815549 355434 browser details YourSeq 71 1028 1107 2000 96.3% chr10 - 59508047 59812327 304281 browser details YourSeq 59 1027 1118 2000 79.8% chr1 - 121544379 121544468 90 browser details YourSeq 58 115 1106 2000 91.5% chr6 - 134734505 135035241 300737 browser details YourSeq 57 59 170 2000 95.5% chr1 - 160873556 160873672 117 browser details YourSeq 54 1036 1110 2000 93.8% chr7 - 118651625 118651700 76 browser details YourSeq 54 1027 1119 2000 84.7% chr1 + 16200930 16201018 89 browser details YourSeq 51 1035 1118 2000 77.8% chr9 - 37915758 37915839 82 browser details YourSeq 48 1027 1110 2000 91.1% chr14 - 81320346 81320428 83 browser details YourSeq 47 71 134 2000 94.5% chr5 - 149055041 149055114 74 browser details YourSeq 47 106 169 2000 82.3% chr11 - 60315193 60315254 62 browser details YourSeq 47 1027 1116 2000 83.7% chr11 - 9336412 9336497 86 browser details YourSeq 47 1035 1109 2000 83.1% chrX + 107428666 107428741 76 browser details YourSeq 46 1027 1108 2000 90.6% chr10 - 109418653 109418733 81 browser details YourSeq 46 1035 1110 2000 92.8% chr11 + 116089419 116089751 333 browser details YourSeq 46 1028 1114 2000 87.4% chr10 + 120507836 120507927 92 browser details YourSeq 45 1035 1110 2000 96.0% chr4 - 104108485 104108561 77 browser details YourSeq 45 1029 1108 2000 90.4% chr1 - 69266780 69266858 79 browser details YourSeq 45 1035 1116 2000 84.4% chr1 - 24044502 24044579 78

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% chr11 - 32251875 32253874 2000 browser details YourSeq 175 214 564 2000 91.2% chr2 + 168964677 168965273 597 browser details YourSeq 175 214 532 2000 90.5% chr17 + 57017011 57017804 794 browser details YourSeq 162 216 564 2000 83.0% chr1 - 180868977 180869248 272 browser details YourSeq 157 214 655 2000 81.4% chr11 - 106706885 106707109 225 browser details YourSeq 157 214 426 2000 92.5% chr9 + 83757608 83757826 219 browser details YourSeq 154 214 412 2000 87.5% chr10 - 60241833 60242018 186 browser details YourSeq 154 214 415 2000 87.9% chr13 + 97532362 97532556 195 browser details YourSeq 149 214 415 2000 86.1% chr11 - 106902913 106903104 192 browser details YourSeq 148 215 393 2000 90.4% chr8 - 86700319 86700496 178 browser details YourSeq 148 210 393 2000 90.8% chr2 - 92134954 92135343 390 browser details YourSeq 148 214 383 2000 93.6% chr17 + 24553710 24553879 170 browser details YourSeq 146 214 417 2000 85.3% chr15 - 93484849 93485036 188 browser details YourSeq 145 214 393 2000 90.6% chr6 - 83675171 83675353 183 browser details YourSeq 145 206 391 2000 89.7% chr2 - 54124574 54124759 186 browser details YourSeq 145 214 389 2000 91.5% chr12 - 83515229 83515407 179 browser details YourSeq 145 214 394 2000 88.9% chr11 - 78192201 78192380 180 browser details YourSeq 145 213 404 2000 88.5% chr15 + 83566646 83566849 204 browser details YourSeq 144 214 389 2000 89.8% chr17 - 83579249 83579423 175 browser details YourSeq 144 215 393 2000 90.5% chr11 - 68478236 68478417 182

Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Nprl3 nitrogen permease regulator-like 3 [ Mus musculus (house mouse) ] Gene ID: 17168, updated on 12-Aug-2019

Gene summary

Official Symbol Nprl3 provided by MGI Official Full Name nitrogen permease regulator-like 3 provided by MGI Primary source MGI:MGI:109258 See related Ensembl:ENSMUSG00000020289 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 Aag; Phg; Mare; HS-26; HS-40; Prox1; CGTHBA; m(alpha)RE Expression Ubiquitous expression in adrenal adult (RPKM 39.2), ovary adult (RPKM 30.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 11 A4; 11 18.83 cM See Nprl3 in Genome Data Viewer Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (32231963..32267707, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (32132419..32167614, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 16 transcripts

Gene: Nprl3 ENSMUSG00000020289

Description nitrogen permease regulator-like 3 [Source:MGI Symbol;Acc:MGI:109258] Gene Synonyms -14 gene, HS-26, HS-40, Mare, Phg, Prox1, m(alpha)RE Location Chromosome 11: 32,225,628-32,267,707 reverse strand. GRCm38:CM001004.2 About this gene This gene has 16 transcripts (splice variants), 201 orthologues, is a member of 1 Ensembl protein family and is associated with 36 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nprl3- ENSMUST00000020530.11 2865 569aa ENSMUSP00000020530.5 Protein coding CCDS24521 Q8VIJ8 TSL:1 201 GENCODE basic APPRIS P1

Nprl3- ENSMUST00000109389.8 1642 544aa ENSMUSP00000105016.2 Protein coding - A7M7S2 TSL:5 202 GENCODE basic

Nprl3- ENSMUST00000129010.1 623 182aa ENSMUSP00000123219.1 Protein coding - A2AAX8 CDS 3' 208 incomplete TSL:3

Nprl3- ENSMUST00000141859.7 3417 77aa ENSMUSP00000120341.1 Nonsense mediated - F2Z3Y4 TSL:1 213 decay

Nprl3- ENSMUST00000136903.7 2945 58aa ENSMUSP00000114781.1 Nonsense mediated - F2Z404 TSL:5 211 decay

Nprl3- ENSMUST00000124640.7 2696 69aa ENSMUSP00000122085.1 Nonsense mediated - F2Z3V7 TSL:1 205 decay

Nprl3- ENSMUST00000137950.7 1602 69aa ENSMUSP00000115594.1 Nonsense mediated - F2Z3V7 TSL:5 212 decay

Nprl3- ENSMUST00000149526.1 537 80aa ENSMUSP00000122231.1 Nonsense mediated - D6RGB2 TSL:5 216 decay

Nprl3- ENSMUST00000129573.1 762 No - Retained intron - - TSL:3 209 protein

Nprl3- ENSMUST00000146890.7 713 No - Retained intron - - TSL:3 214 protein

Nprl3- ENSMUST00000125256.1 705 No - Retained intron - - TSL:3 206 protein

Nprl3- ENSMUST00000148636.1 680 No - Retained intron - - TSL:2 215 protein

Nprl3- ENSMUST00000109390.7 3531 No - lncRNA - - TSL:1 203 protein

Nprl3- ENSMUST00000127657.1 602 No - lncRNA - - TSL:3 207 protein

Nprl3- ENSMUST00000123411.1 411 No - lncRNA - - TSL:3 204 protein

Nprl3- ENSMUST00000132856.1 406 No - lncRNA - - TSL:2 210 protein

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62.08 kb Forward strand 32.22Mb 32.23Mb 32.24Mb 32.25Mb 32.26Mb 32.27Mb Genes Mpg-203 >nonsense mediated decay Hba-x-201 >protein coding (Comprehensive set...

Mpg-201 >protein coding Hba-x-202 >protein coding

Mpg-204 >retained intron

Mpg-202 >nonsense mediated decay

Contigs AL929446.5 > AL662780.20 > Genes (Comprehensive set... < Rhbdf1-201protein coding < Nprl3-205nonsense mediated decay

< Rhbdf1-205retained intron < Nprl3-213nonsense mediated decay

< Rhbdf1-208nonsense mediated decay < Nprl3-203lncRNA < Nprl3-210lncRNA

< Rhbdf1-202nonsense mediated decay < Nprl3-201protein coding

< Rhbdf1-209protein coding < Nprl3-211nonsense mediated decay

< Rhbdf1-207protein coding < Nprl3-215retained intron< Nprl3-204lncRNA

< Nprl3-212nonsense mediated decay

< Nprl3-202protein coding

< Nprl3-206retained intron < Nprl3-207lncRNA

< Nprl3-209retained intron < Nprl3-208protein coding

< Nprl3-214retained intron

< Nprl3-216nonsense mediated decay

Regulatory Build

32.22Mb 32.23Mb 32.24Mb 32.25Mb 32.26Mb 32.27Mb Reverse strand 62.08 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000020530

< Nprl3-201protein coding

Reverse strand 35.74 kb

ENSMUSP00000020... MobiDB lite Low complexity (Seg) Superfamily Galactose-binding-like domain superfamily Pfam Nitrogen permease regulator 3 PANTHER Nitrogen permease regulator 3

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant splice region variant stop retained variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 569

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

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