Mouse Ern1 Conditional Knockout Project (CRISPR/Cas9)

http://www.alphaknockout.com/ Mouse Ern1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ern1 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ern1 gene ( NCBI Reference Sequence: NM_023913 ; Ensembl: ENSMUSG00000020715 ) is located on mouse chromosome 11. 22 exons are identified , with the ATG start codon in exon 1 and the TGA stop codon in exon 22 (Transcript: ENSMUST00000001059). 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 Ern1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-386O11 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: Mice homozygous for null mutations display embryonic lethality during organogenesis. Homozygous mice may also display decreased embryo size, impaired hematopoiesis, and/or placental abnormalities.

The knockout of Exon 2 will result in frameshift of the gene, and covers 4.13% of the coding region. The size of intron 1 for 5'-loxP site insertion: 28588 bp, and the size of intron 2 for 3'-loxP site insertion: 21784 bp. The size of effective cKO region: ~1620 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 22 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ern1 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(7121bp) | A(26.63% 1896) | C(19.59% 1395) | G(22.29% 1587) | T(31.5% 2243)

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 - 106459279 106462278 3000 browser details YourSeq 213 2505 2885 3000 90.3% chr4 + 132903419 132903766 348 browser details YourSeq 197 2503 2886 3000 89.9% chr16 + 18438940 18439256 317 browser details YourSeq 190 2677 2902 3000 95.8% chr5 - 121456971 121457210 240 browser details YourSeq 190 2526 2885 3000 96.7% chr11 - 96990726 96991258 533 browser details YourSeq 190 2672 2885 3000 93.0% chr4 + 123761529 123761729 201 browser details YourSeq 187 2678 2887 3000 97.5% chr6 - 34898766 34898981 216 browser details YourSeq 187 2676 2885 3000 96.6% chr3 - 121963381 121963591 211 browser details YourSeq 187 2683 2882 3000 96.0% chr2 + 97575334 97575532 199 browser details YourSeq 187 2673 2884 3000 97.0% chr18 + 4058595 4058813 219 browser details YourSeq 186 2681 2885 3000 98.5% chr12 - 111472406 111472617 212 browser details YourSeq 186 2677 2901 3000 95.2% chr17 + 22366064 22366288 225 browser details YourSeq 185 2678 2885 3000 93.5% chr8 - 117428076 117428273 198 browser details YourSeq 185 2678 2884 3000 97.5% chr15 - 82751079 82751295 217 browser details YourSeq 185 2678 2884 3000 93.9% chr19 + 53763673 53763868 196 browser details YourSeq 184 2678 2884 3000 97.5% chrX - 82253604 82253817 214 browser details YourSeq 184 2678 2884 3000 97.5% chr9 - 56245554 56245768 215 browser details YourSeq 184 2695 2901 3000 96.1% chr9 - 20392946 20393154 209 browser details YourSeq 184 2678 2885 3000 97.5% chr7 - 28707963 28708205 243 browser details YourSeq 184 2682 2885 3000 98.0% chr17 - 70359064 70359273 210

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% chr11 - 106455658 106458657 3000 browser details YourSeq 255 375 694 3000 91.3% chr2 + 128725450 128725782 333 browser details YourSeq 247 375 698 3000 93.4% chr3 + 57615221 57615587 367 browser details YourSeq 246 373 689 3000 87.6% chr7 + 110783773 110784078 306 browser details YourSeq 245 378 692 3000 90.5% chr8 - 22473861 22474184 324 browser details YourSeq 244 367 694 3000 88.8% chr15 - 67511609 67511946 338 browser details YourSeq 243 378 711 3000 91.3% chr5 - 120765402 120765756 355 browser details YourSeq 243 378 690 3000 91.4% chr8 + 117289757 117290091 335 browser details YourSeq 243 375 689 3000 92.7% chr3 + 97218513 97218851 339 browser details YourSeq 242 369 715 3000 90.9% chr16 + 32450411 32450775 365 browser details YourSeq 241 378 694 3000 93.3% chrX - 153677965 153678311 347 browser details YourSeq 241 377 694 3000 86.9% chr17 - 74505541 74505848 308 browser details YourSeq 241 375 694 3000 90.8% chr13 - 8997205 8997727 523 browser details YourSeq 241 375 706 3000 89.6% chrX + 94024090 94024414 325 browser details YourSeq 241 375 694 3000 90.9% chr3 + 103162503 103162863 361 browser details YourSeq 240 376 694 3000 89.7% chr9 - 75433618 75433965 348 browser details YourSeq 240 375 676 3000 91.4% chr16 - 11163008 11163318 311 browser details YourSeq 240 377 694 3000 88.3% chr15 - 83554404 83554716 313 browser details YourSeq 240 379 690 3000 89.9% chr14 + 20353783 20354089 307 browser details YourSeq 239 376 690 3000 88.0% chr18 - 64880049 64880356 308

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

Page 4 of 7 http://www.alphaknockout.com/ Gene and protein information: Ern1 endoplasmic reticulum (ER) to nucleus signalling 1 [ Mus musculus (house mouse) ] Gene ID: 78943, updated on 10-Jan-2021

Gene summary

Official Symbol Ern1 provided by MGI Official Full Name endoplasmic reticulum (ER) to nucleus signalling 1 provided by MGI Primary source MGI:MGI:1930134 See related Ensembl:ENSMUSG00000020715 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Ir; Ire; Ire1a; Ire1p; C85377; AI225830; Ire1alpha; 9030414B18Rik Expression Broad expression in adrenal adult (RPKM 76.6), ovary adult (RPKM 25.1) and 15 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 E1 See Ern1 in Genome Data Viewer

Exon count: 22

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) 11 NC_000077.7 (106285474..106378701, complement)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (106397620..106487796, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (106258934..106349110, complement)

Chromosome 11 - NC_000077.7

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Transcript information: This gene has 5 transcripts

Gene: Ern1 ENSMUSG00000020715

Description endoplasmic reticulum (ER) to nucleus signalling 1 [Source:MGI Symbol;Acc:MGI:1930134] Gene Synonyms 9030414B18Rik, Ire1a, Ire1alpha, Ire1p Location Chromosome 11: 106,394,650-106,487,852 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 195 orthologues, 1 paralogue and is associated with 15 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Match Flags

Ern1- ENSMUST00000001059.8 6946 977aa ENSMUSP00000001059.2 Protein coding CCDS25556 Q9EQY0 TSL:1 201 GENCODE basic APPRIS P1

Ern1- ENSMUST00000106801.7 1957 408aa ENSMUSP00000102413.1 Protein coding - Q3TBZ5 TSL:1 204 Q9EQY0 GENCODE basic

Ern1- ENSMUST00000106799.1 660 66aa ENSMUSP00000102411.1 Protein coding - B1ATQ7 TSL:1 202 GENCODE basic

Ern1- ENSMUST00000106800.1 654 118aa ENSMUSP00000102412.1 Protein coding - B1ARJ5 TSL:3 203 GENCODE basic

Ern1- ENSMUST00000131895.1 3354 No - Processed - - TSL:5 205 protein transcript

113.20 kb Forward strand

106.40Mb 106.42Mb 106.44Mb 106.46Mb 106.48Mb Contigs AL604045.7 > AL663053.12 > Genes (Comprehensive set... < Icam2-203protein coding < Ern1-204protein coding

< Icam2-204protein coding < Ern1-203protein coding

< Ern1-201protein coding

< Ern1-205processed transcript

< Ern1-202protein coding

Regulatory Build

106.40Mb 106.42Mb 106.44Mb 106.46Mb 106.48Mb Reverse strand 113.20 kb

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

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Transcript: ENSMUST00000001059

< Ern1-201protein coding

Reverse strand 93.15 kb

ENSMUSP00000001... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Quinoprotein alcohol dehydrogenase-like superfamily Protein kinase-like domain superfamily

SMART Pyrrolo-quinoline quinone beta-propeller repeat Protein kinase domain PUB domain

Pfam Protein kinase domain KEN domain

PROSITE profiles Protein kinase domain KEN domain

PROSITE patterns Serine/threonine-protein kinase, active site PANTHER PTHR13954

PTHR13954:SF17 Gene3D WD40/YVTN repeat-like-containing domain superfamily 1.10.510.10 KEN domain superfamily

3.30.200.20 CDD cd09769 cd13982 cd10422

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

Variant Legend missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 977

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

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