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Mouse Nbr1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Nbr1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nbr1 (NCBI Reference Sequence: NM_001252220 ; Ensembl: ENSMUSG00000017119 ) is located on Mouse 11. 24 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 24 (Transcript: ENSMUST00000103099). Exon 8~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nbr1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-281H12 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: Homozygous mice of the genetic truncation allele had an age-dependent increase in mass and bone mineral density. Mice homozygous for a floxed allele activated in T cells exhibit decreased ovalbumin-induced inflammation and defective Th2 polarization.

Exon 8 starts from about 7.02% of the coding region. The knockout of Exon 8~10 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 2309 bp, and the size of intron 10 for 3'-loxP site insertion: 684 bp. The size of effective cKO region: ~2282 bp. The cKO 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 8 9 10 11 12 24 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Nbr1 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(8782bp) | A(29.91% 2627) | C(20.84% 1830) | T(27.48% 2413) | G(21.77% 1912)

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% chr11 + 101561380 101564379 3000 browser details YourSeq 248 2647 2999 3000 89.3% chr2 + 153507624 153507967 344 browser details YourSeq 236 2648 2971 3000 92.2% chr9 - 40796738 40797093 356 browser details YourSeq 232 2633 2972 3000 90.9% chr10 - 117314756 117315167 412 browser details YourSeq 229 2648 2964 3000 91.7% chr1_GL456212_random - 33050 33470 421 browser details YourSeq 229 2648 2964 3000 91.7% chr1 - 85315430 85315850 421 browser details YourSeq 226 2675 2974 3000 91.0% chr3 - 41441643 41441985 343 browser details YourSeq 226 2656 2964 3000 90.1% chr1 - 153496630 153497048 419 browser details YourSeq 212 2649 2925 3000 91.5% chr10 - 81967273 81967699 427 browser details YourSeq 210 2676 2973 3000 91.5% chr2 - 132616294 132616961 668 browser details YourSeq 207 2648 2925 3000 90.0% chr9 - 88845882 88846218 337 browser details YourSeq 200 2669 2925 3000 92.1% chr8 - 72291923 72292204 282 browser details YourSeq 199 2656 2921 3000 90.7% chr11 - 86933916 87466845 532930 browser details YourSeq 199 2648 2920 3000 89.4% chr1 + 60145665 60145980 316 browser details YourSeq 182 2674 2925 3000 90.7% chr11 + 80358451 80358805 355 browser details YourSeq 177 2681 2922 3000 91.2% chr11 + 69659436 69659696 261 browser details YourSeq 176 2687 2925 3000 91.2% chr19 + 6379507 6379765 259 browser details YourSeq 171 2656 2924 3000 89.9% chr7 + 45099244 45099593 350 browser details YourSeq 170 2737 2975 3000 90.6% chr11 - 115774278 115774540 263 browser details YourSeq 148 2656 2977 3000 90.8% chr2 - 30085700 30086123 424

Note: The 3000 bp section upstream of Exon 8 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% chr11 + 101566662 101569661 3000 browser details YourSeq 299 187 896 3000 91.5% chr4 - 94653621 94654261 641 browser details YourSeq 276 207 891 3000 89.3% chr1 - 58575249 58575570 322 browser details YourSeq 276 203 892 3000 88.8% chr7 + 141121782 141122121 340 browser details YourSeq 262 187 897 3000 89.8% chr18 - 38237530 38237919 390 browser details YourSeq 262 127 783 3000 90.3% chr9 + 56902610 56903087 478 browser details YourSeq 261 127 782 3000 89.3% chr2 - 154653662 154653971 310 browser details YourSeq 260 128 783 3000 90.0% chr17 - 27044604 27044896 293 browser details YourSeq 260 128 779 3000 91.8% chr14 + 20896630 20896939 310 browser details YourSeq 254 687 1209 3000 93.8% chr16 + 13873615 13874218 604 browser details YourSeq 253 128 778 3000 92.1% chr10 + 76083891 76084504 614 browser details YourSeq 236 241 869 3000 89.0% chr13 - 23616065 23616323 259 browser details YourSeq 226 128 758 3000 90.6% chr11 + 119290075 119290684 610 browser details YourSeq 220 128 780 3000 88.5% chr16 + 32029529 32029872 344 browser details YourSeq 217 687 1245 3000 90.0% chr12 + 16923000 16923264 265 browser details YourSeq 211 688 1139 3000 89.8% chr16 - 18699110 18699381 272 browser details YourSeq 210 128 776 3000 89.8% chr2 - 33656109 33656429 321 browser details YourSeq 204 127 778 3000 86.6% chr16 - 17221879 17222177 299 browser details YourSeq 204 688 897 3000 98.6% chr14 - 32328287 32328496 210 browser details YourSeq 204 686 897 3000 98.6% chr3 + 153808892 153809108 217

Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Nbr1 NBR1, autophagy cargo receptor [ Mus musculus () ] Gene ID: 17966, updated on 12-Aug-2019

Gene summary

Official Symbol Nbr1 provided by MGI Official Full Name NBR1, autophagy cargo receptor provided by MGI Primary source MGI:MGI:108498 See related Ensembl:ENSMUSG00000017119 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 mKIAA0049 Expression Ubiquitous expression in testis adult (RPKM 73.0), placenta adult (RPKM 24.7) and 28 other tissues See more Orthologs all

Genomic context

Location: 11 D; 11 65.36 cM See Nbr1 in Genome Data Viewer

Exon count: 25

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (101552107..101581951)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (101414172..101443259)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 21 transcripts

Gene: Nbr1 ENSMUSG00000017119

Description NBR1, autophagy cargo receptor [Source:MGI Symbol;Acc:MGI:108498] Location Chromosome 11: 101,552,149-101,581,951 forward strand. GRCm38:CM001004.2 About this gene This gene has 21 transcripts (splice variants), 232 orthologues, is a member of 1 Ensembl protein family and is associated with 18 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nbr1- ENSMUST00000103099.7 4789 988aa ENSMUSP00000099388.1 Protein coding CCDS25475 A1L329 TSL:1 203 P97432 GENCODE basic APPRIS P3

Nbr1- ENSMUST00000103098.8 4654 988aa ENSMUSP00000099387.2 Protein coding CCDS25475 A1L329 TSL:1 202 P97432 GENCODE basic APPRIS P3

Nbr1- ENSMUST00000107208.7 4344 906aa ENSMUSP00000102826.1 Protein coding CCDS56810 A2A4N5 TSL:1 204 GENCODE basic

Nbr1- ENSMUST00000107213.7 4339 951aa ENSMUSP00000102831.1 Protein coding CCDS56811 A2A4N8 TSL:1 206 GENCODE basic APPRIS ALT2

Nbr1- ENSMUST00000107218.9 3353 988aa ENSMUSP00000102836.3 Protein coding CCDS25475 A1L329 TSL:5 207 P97432 GENCODE basic APPRIS P3

Nbr1- ENSMUST00000107212.7 4168 963aa ENSMUSP00000102830.1 Protein coding - A2A4N7 TSL:5 205 GENCODE basic APPRIS ALT2

Nbr1- ENSMUST00000149019.1 3330 712aa ENSMUSP00000119900.1 Protein coding - F6S397 CDS 5' 217 incomplete TSL:1

Nbr1- ENSMUST00000071537.12 3239 988aa ENSMUSP00000071467.6 Protein coding - K3W4P1 TSL:5 201 GENCODE basic APPRIS ALT2

Nbr1- ENSMUST00000147239.7 563 43aa ENSMUSP00000122097.1 Protein coding - A2A4R0 CDS 3' 215 incomplete TSL:5

Nbr1- ENSMUST00000127421.7 265 43aa ENSMUSP00000121628.1 Protein coding - A2A4R0 CDS 3' 209 incomplete TSL:3

Nbr1- ENSMUST00000123558.7 4392 888aa ENSMUSP00000133619.1 Nonsense mediated - Q05BC8 TSL:1 208 decay

Nbr1- ENSMUST00000136185.1 276 41aa ENSMUSP00000134500.1 Nonsense mediated - G3UZH6 TSL:3 211 decay

Nbr1- ENSMUST00000146452.1 810 No - Retained intron - - TSL:3 214 protein

Nbr1- ENSMUST00000172744.1 804 No - Retained intron - - TSL:3 219 protein

Nbr1- ENSMUST00000148805.1 639 No - Retained intron - - TSL:2 216 protein

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Nbr1- ENSMUST00000127871.1 594 No - Retained intron - - TSL:2 210 protein

Nbr1- ENSMUST00000174013.1 463 No - Retained intron - - TSL:3 220 protein

Nbr1- ENSMUST00000141170.1 418 No - Retained intron - - TSL:2 212 protein

Nbr1- ENSMUST00000144517.1 364 No - lncRNA - - TSL:2 213 protein

Nbr1- ENSMUST00000149170.1 342 No - lncRNA - - TSL:3 218 protein

Nbr1- ENSMUST00000184092.1 63 No - misc RNA - - TSL:NA 221 protein

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49.80 kb Forward strand 101.55Mb 101.56Mb 101.57Mb 101.58Mb 101.59Mb (Comprehensive set... Nbr1-203 >protein coding Tmem106a-202 >protein coding

Nbr1-215 >protein coding Nbr1-214 >retained intron Nbr1-219 >retained intron Tmem106a-201 >protein coding

Nbr1-202 >protein coding Tmem106a-206 >lncRNA

Nbr1-204 >protein coding Tmem106a-203 >protein coding

Nbr1-208 >nonsense mediated decay Tmem106a-204 >protein coding

Nbr1-206 >protein coding Tmem106a-205 >lncRNA

Nbr1-218 >lncRNA Nbr1-213 >lncRNA Nbr1-216 >retained intron

Nbr1-205 >protein coding

Nbr1-209 >protein coding Nbr1-210 >retained intron Nbr1-212 >retained intron

Nbr1-211 >nonsense mediated decay Nbr1-217 >protein coding

Nbr1-207 >protein coding

Nbr1-201 >protein coding

Nbr1-221 >misc RNA Nbr1-220 >retained intron

Contigs AL590996.12 > AL590994.13 > Genes < Brca1-201protein coding (Comprehensive set...

< Brca1-203nonsense mediated decay

< Brca1-206protein coding

Regulatory Build

101.55Mb 101.56Mb 101.57Mb 101.58Mb 101.59Mb Reverse strand 49.80 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

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Transcript: ENSMUST00000103099

29.80 kb Forward strand

Nbr1-203 >protein coding

ENSMUSP00000099... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF54277 SSF57850 UBA-like superfamily

SMART PB1 domain Zinc finger, ZZ-type

Pfam PB1 domain Zinc finger, ZZ-type Next to BRCA1, central domain

PROSITE profiles PB1 domain Zinc finger, ZZ-type Ubiquitin-associated domain

PROSITE patterns Zinc finger, ZZ-type PANTHER Next to BRCA1 gene 1 protein

PTHR20930 Gene3D 3.10.20.90 3.30.60.90 Immunoglobulin-like fold 1.10.8.10

CDD NBR1, PB1 domain cd02340 Next to BRCA1, central domain cd14319

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

Variant Legend inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 988

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

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