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Mouse Add2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Add2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Add2 gene (NCBI Reference Sequence: NM_001271858 ; Ensembl: ENSMUSG00000030000 ) is located on Mouse chromosome 6. 16 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000204059). 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 Add2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-434N19 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 targeted mutations that inactivate the gene display mild anemia with compensated hemolysis, marked alteration in osmotic fragility, predominant presence of elliptocytes in the blood and increased blood pressure.

Exon 3 starts from about 100% 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: 3014 bp, and the size of intron 3 for 3'-loxP site insertion: 859 bp. The size of effective cKO region: ~683 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 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Add2 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(7183bp) | A(25.11% 1804) | C(22.39% 1608) | T(28.71% 2062) | G(23.79% 1709)

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% chr6 + 86082613 86085612 3000 browser details YourSeq 59 1754 2006 3000 89.4% chr17 - 34890656 35217291 326636 browser details YourSeq 53 1632 1795 3000 98.3% chr5 + 127422160 127422417 258 browser details YourSeq 43 1754 1842 3000 76.0% chr4 - 155667394 155667467 74 browser details YourSeq 38 1756 1819 3000 95.3% chr6 - 127060862 127060926 65 browser details YourSeq 37 1754 1819 3000 93.1% chr4 + 43576382 43576448 67 browser details YourSeq 35 1754 1817 3000 92.7% chr2 - 73461388 73461452 65 browser details YourSeq 35 1754 1817 3000 92.7% chr11 + 117684400 117684464 65 browser details YourSeq 34 1127 1164 3000 97.4% chr15 - 34295201 34295515 315 browser details YourSeq 34 1754 1817 3000 94.8% chr17 + 23875154 23875218 65 browser details YourSeq 32 1754 1817 3000 94.5% chr4 - 105050408 105050472 65 browser details YourSeq 30 1762 1817 3000 94.2% chr15 - 31544435 31544491 57 browser details YourSeq 28 1765 1817 3000 93.8% chr1 - 43308499 43308551 53 browser details YourSeq 27 1765 1817 3000 93.6% chr6 + 38733636 38733689 54 browser details YourSeq 27 1765 1817 3000 93.6% chr18 + 60642596 60642649 54 browser details YourSeq 24 896 924 3000 81.5% chr16 - 35392555 35392581 27 browser details YourSeq 21 2817 2847 3000 83.9% chr13 + 48072391 48072421 31 browser details YourSeq 21 2243 2269 3000 88.9% chr10 + 52470271 52470297 27

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% chr6 + 86086296 86089295 3000 browser details YourSeq 80 2516 2681 3000 76.4% chr14 + 32238087 32238227 141 browser details YourSeq 64 2572 2688 3000 92.2% chr11 + 83280279 83280474 196 browser details YourSeq 53 2541 2687 3000 95.0% chr5 + 24822737 24822973 237 browser details YourSeq 47 2282 2688 3000 62.3% chr15 + 86006563 86006646 84 browser details YourSeq 44 2606 2680 3000 75.4% chrX - 134236227 134236299 73 browser details YourSeq 43 2542 2688 3000 64.4% chr11 - 58338703 58338812 110 browser details YourSeq 42 2539 2636 3000 93.9% chr1 + 174611768 174612019 252 browser details YourSeq 41 2574 2636 3000 79.1% chr1 - 188912921 188912969 49 browser details YourSeq 41 2771 2904 3000 91.9% chr1 + 53028334 53028468 135 browser details YourSeq 40 2525 2688 3000 68.2% chr1 - 182061313 182061428 116 browser details YourSeq 39 2584 2684 3000 84.3% chr11 - 88791277 88791594 318 browser details YourSeq 39 2572 2634 3000 91.5% chr1 + 167314355 167314453 99 browser details YourSeq 39 2610 2732 3000 95.4% chr1 + 56456368 56456526 159 browser details YourSeq 38 2629 2686 3000 82.8% chr3 - 35728817 35728874 58 browser details YourSeq 38 2640 2732 3000 91.4% chr11 - 106773244 106773338 95 browser details YourSeq 38 2639 2688 3000 88.0% chr8 + 106520119 106520168 50 browser details YourSeq 37 2621 2684 3000 82.5% chr10 + 111075993 111076061 69 browser details YourSeq 36 2572 2634 3000 95.0% chr12 - 111023165 111023249 85 browser details YourSeq 36 2539 2680 3000 82.5% chr11 - 94323556 94323830 275

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: Add2 adducin 2 (beta) [ Mus musculus (house mouse) ] Gene ID: 11519, updated on 10-Oct-2019

Gene summary

Official Symbol Add2 provided by MGI Official Full Name adducin 2 (beta) provided by MGI Primary source MGI:MGI:87919 See related Ensembl:ENSMUSG00000030000 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as add97; 2900072M03Rik Summary This gene encodes the beta subunit of the adducin family. Adducins, encoded by alpha, beta and gamma genes, are Expression heteromeric proteins that crosslink actin filaments with spectrin at the cytoskeletal membrane. This protein, primarily found in the brain and hematopoietic cells, is regulated by phosphorylation and calmodulin interactions as it promotes spectrin assembly onto actin filaments, bundles actin and caps barbed ends of actin filaments. In mouse, deficiency of this gene can lead to mild hemolytic anemia and impaired synaptic plasticity. Mutations of this gene in mouse serve as a pathophysiological model for hereditary spherocytosis and hereditary elliptocytosis. Alternative splicing results in multiple transcript variants that encode different protein isoforms. [provided by RefSeq, Dec 2012] Orthologs Biased expression in CNS E18 (RPKM 33.1), whole brain E14.5 (RPKM 28.0) and 10 other tissues See more human all

Genomic context

Location: 6 C3-D1; 6 37.55 cM See Add2 in Genome Data Viewer Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (86028681..86124409)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (86028078..86069549)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 10 transcripts

Gene: Add2 ENSMUSG00000030000

Description adducin 2 (beta) [Source:MGI Symbol;Acc:MGI:87919] Gene Synonyms 2900072M03Rik Location Chromosome 6: 86,028,681-86,124,409 forward strand. GRCm38:CM000999.2 About this gene This gene has 10 transcripts (splice variants), 199 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 32 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Add2-209 ENSMUST00000204059.2 8182 725aa ENSMUSP00000145160.1 Protein coding CCDS20308 Q9QYB8 TSL:1 GENCODE basic APPRIS P3

Add2-204 ENSMUST00000203366.2 3407 562aa ENSMUSP00000144849.1 Protein coding CCDS85085 Q9QYB8 TSL:5 GENCODE basic APPRIS ALT2

Add2-210 ENSMUST00000205034.2 3266 562aa ENSMUSP00000145034.1 Protein coding CCDS85085 Q9QYB8 TSL:1 GENCODE basic APPRIS ALT2

Add2-207 ENSMUST00000203724.2 3143 725aa ENSMUSP00000145296.1 Protein coding CCDS20308 Q9QYB8 TSL:1 GENCODE basic APPRIS P3

Add2-201 ENSMUST00000032069.7 3119 725aa ENSMUSP00000032069.5 Protein coding CCDS20308 Q9QYB8 TSL:1 GENCODE basic APPRIS P3

Add2-208 ENSMUST00000203786.2 2289 725aa ENSMUSP00000144694.1 Protein coding CCDS20308 Q9QYB8 TSL:1 GENCODE basic APPRIS P3

Add2-202 ENSMUST00000203196.2 1970 562aa ENSMUSP00000145104.1 Protein coding CCDS85085 Q9QYB8 TSL:1 GENCODE basic APPRIS ALT2

Add2-203 ENSMUST00000203279.1 1454 477aa ENSMUSP00000145452.1 Protein coding - Q9QYB8 TSL:2 GENCODE basic

Add2-205 ENSMUST00000203445.2 779 184aa ENSMUSP00000145494.1 Protein coding - A0A0N4SWF0 CDS 3' incomplete TSL:3

Add2-206 ENSMUST00000203529.2 1408 No protein - Retained intron - - TSL:1

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115.73 kb Forward strand 86.02Mb 86.04Mb 86.06Mb 86.08Mb 86.10Mb 86.12Mb Genes (Comprehensive set... Figla-202 >lncRNA Gm44090-201 >TEC Add2-207 >protein coding

Figla-201 >protein coding Add2-205 >protein coding

Add2-206 >retained intron

Add2-209 >protein coding

Add2-204 >protein coding

Add2-210 >protein coding

Add2-201 >protein coding

Add2-208 >protein coding

Add2-202 >protein coding

Add2-203 >protein coding

Contigs < AC158679.8 AC158645.6 >

Genes < Gm44089-201lncRNA (Comprehensive set...

Regulatory Build

86.02Mb 86.04Mb 86.06Mb 86.08Mb 86.10Mb 86.12Mb Reverse strand 115.73 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000204059

95.71 kb Forward strand

Add2-209 >protein coding

ENSMUSP00000145... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Class II aldolase/adducin N-terminal domain superfamily SMART Class II aldolase/adducin N-terminal Pfam Class II aldolase/adducin N-terminal PANTHER PTHR10672

Beta-adducin Gene3D Class II aldolase/adducin N-terminal domain superfamily CDD cd00398

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

Variant Legend missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 725

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

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