https://www.alphaknockout.com

Mouse Add3 Knockout Project (CRISPR/Cas9)

Objective: To create a Add3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Add3 gene (NCBI Reference Sequence: NM_001164099 ; Ensembl: ENSMUSG00000025026 ) is located on Mouse chromosome 19. 15 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000237301). Exon 4~12 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: Mice homozygous for a knock-out allele exhibit normal blood pressure and show no significant alterations in red blood cell or platelet structure and function.

Exon 4 starts from about 15.82% of the coding region. Exon 4~12 covers 60.15% of the coding region. The size of effective KO region: ~8314 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 4 5 6 7 8 9 10 11 12 15

Legends Exon of mouse Add3 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 4 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

Overview of the Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 12 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(31.2% 624) | C(16.6% 332) | T(28.5% 570) | G(23.7% 474)

Note: The 2000 bp section upstream 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(34.1% 682) | C(20.05% 401) | T(23.6% 472) | G(22.25% 445)

Note: The 2000 bp section downstream of Exon 12 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% chr19 + 53229181 53231180 2000 browser details YourSeq 166 765 1225 2000 93.8% chr11 - 80440776 80441277 502 browser details YourSeq 165 702 1215 2000 94.2% chr15 + 73412020 73412623 604 browser details YourSeq 159 673 1230 2000 85.5% chr11 - 30390411 30390931 521 browser details YourSeq 159 1050 1235 2000 91.9% chr10 - 14592835 14593010 176 browser details YourSeq 158 1050 1236 2000 92.4% chr15 + 100044332 100044508 177 browser details YourSeq 157 1050 1230 2000 93.9% chr14 - 48633789 48633983 195 browser details YourSeq 157 978 1225 2000 93.9% chr11 - 70794457 70794846 390 browser details YourSeq 157 1060 1595 2000 85.7% chr1 + 146541945 146542218 274 browser details YourSeq 156 1050 1225 2000 95.4% chr13 - 74288407 74288582 176 browser details YourSeq 156 1050 1236 2000 95.5% chr11 - 76062089 76062280 192 browser details YourSeq 156 1050 1318 2000 93.9% chr4 + 140200940 140201293 354 browser details YourSeq 156 703 1210 2000 93.4% chr17 + 15567742 15568294 553 browser details YourSeq 154 1050 1225 2000 94.8% chr2 - 3461452 3461627 176 browser details YourSeq 154 1050 1230 2000 93.6% chr17 - 45467056 45467235 180 browser details YourSeq 154 1050 1235 2000 92.7% chr1 - 74581938 74582112 175 browser details YourSeq 154 1050 1224 2000 95.9% chr1 + 43697560 43697734 175 browser details YourSeq 153 1050 1224 2000 94.8% chr11 - 79000660 79000834 175 browser details YourSeq 153 1050 1224 2000 95.9% chr9 + 103200775 103200949 175 browser details YourSeq 153 1050 1234 2000 94.8% chr5 + 149475825 149476010 186

Note: The 2000 bp section upstream of Exon 4 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% chr19 + 53239495 53241494 2000 browser details YourSeq 173 892 1189 2000 94.0% chr12 - 71935834 71936249 416 browser details YourSeq 149 1000 1177 2000 94.8% chr16 + 84768402 84768598 197 browser details YourSeq 147 1007 1215 2000 94.0% chr5 - 30352471 30352768 298 browser details YourSeq 144 1001 1191 2000 88.4% chr4 - 44296321 44296504 184 browser details YourSeq 143 993 1394 2000 82.8% chr13 - 63195981 63196157 177 browser details YourSeq 142 1004 1394 2000 84.9% chr2 + 13736700 13736877 178 browser details YourSeq 140 1002 1394 2000 83.3% chr2 - 123254263 123254438 176 browser details YourSeq 140 1008 1213 2000 85.8% chr4 + 125280013 125280190 178 browser details YourSeq 138 1002 1394 2000 82.5% chr13 - 69260236 69260403 168 browser details YourSeq 137 799 1155 2000 83.5% chr2 - 28697316 28697494 179 browser details YourSeq 137 1013 1394 2000 93.1% chr1 - 74810586 74811042 457 browser details YourSeq 137 1002 1153 2000 95.4% chr12 + 108618829 108618981 153 browser details YourSeq 137 1001 1155 2000 94.8% chr12 + 15877587 15877741 155 browser details YourSeq 137 1003 1157 2000 93.5% chr10 + 62799179 62799332 154 browser details YourSeq 136 1002 1394 2000 83.0% chr10 - 80516016 80516183 168 browser details YourSeq 134 1005 1157 2000 95.4% chr12 - 109014025 109014177 153 browser details YourSeq 134 984 1150 2000 93.5% chr11 - 100743781 100743956 176 browser details YourSeq 134 1004 1158 2000 94.7% chr2 + 144531896 144532052 157 browser details YourSeq 134 996 1154 2000 92.4% chr11 + 97484169 97484332 164

Note: The 2000 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Add3 adducin 3 (gamma) [ Mus musculus (house mouse) ] Gene ID: 27360, updated on 10-Oct-2019

Gene summary

Official Symbol Add3 provided by MGI Official Full Name adducin 3 (gamma) provided by MGI Primary source MGI:MGI:1351615 See related Ensembl:ENSMUSG00000025026 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 R75380; AI463285 Expression Ubiquitous expression in bladder adult (RPKM 34.1), cerebellum adult (RPKM 18.5) and 27 other tissues See more Orthologs human all

Genomic context

Location: 19 D2; 19 47.18 cM See Add3 in Genome Data Viewer Exon count: 22

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (53140443..53247326)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (53214935..53321889)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 16 transcripts

Gene: Add3 ENSMUSG00000025026

Description adducin 3 (gamma) [Source:MGI Symbol;Acc:MGI:1351615] Location Chromosome 19: 53,140,443-53,247,399 forward strand. GRCm38:CM001012.2 About this gene This gene has 16 transcripts (splice variants), 263 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Add3- ENSMUST00000111741.9 4426 674aa ENSMUSP00000107370.3 Protein coding CCDS50467 Q3UNK1 TSL:1 203 Q9QYB5 GENCODE basic APPRIS ALT1

Add3- ENSMUST00000025999.6 4334 706aa ENSMUSP00000025999.6 Protein coding CCDS29900 Q9QYB5 TSL:1 201 GENCODE basic APPRIS P3

Add3- ENSMUST00000050096.14 4297 674aa ENSMUSP00000052245.7 Protein coding CCDS50467 Q3UNK1 TSL:1 202 Q9QYB5 GENCODE basic APPRIS ALT1

Add3- ENSMUST00000236296.1 4075 706aa ENSMUSP00000157697.1 Protein coding CCDS29900 Q9QYB5 GENCODE basic 207 APPRIS P3

Add3- ENSMUST00000237430.1 4054 706aa ENSMUSP00000157955.1 Protein coding CCDS29900 Q9QYB5 GENCODE basic 213 APPRIS P3

Add3- ENSMUST00000237301.1 4003 706aa ENSMUSP00000158149.1 Protein coding CCDS29900 Q9QYB5 GENCODE basic 212 APPRIS P3

Add3- ENSMUST00000237832.1 2985 674aa ENSMUSP00000157432.1 Protein coding CCDS50467 Q3UNK1 GENCODE basic 215 Q9QYB5 APPRIS ALT1

Add3- ENSMUST00000238130.1 2911 674aa ENSMUSP00000158154.1 Protein coding CCDS50467 Q3UNK1 GENCODE basic 216 Q9QYB5 APPRIS ALT1

Add3- ENSMUST00000235846.1 2852 674aa ENSMUSP00000157970.1 Protein coding CCDS50467 Q3UNK1 GENCODE basic 206 Q9QYB5 APPRIS ALT1

Add3- ENSMUST00000237224.1 2719 674aa ENSMUSP00000158368.1 Protein coding CCDS50467 Q3UNK1 GENCODE basic 211 Q9QYB5 APPRIS ALT1

Add3- ENSMUST00000235754.1 2967 700aa ENSMUSP00000157677.1 Protein coding - A0A494B9K4 GENCODE basic 205

Add3- ENSMUST00000237099.1 817 189aa ENSMUSP00000158231.1 Protein coding - A0A494BAX9 CDS 3' 209 incomplete

Add3- ENSMUST00000235133.1 473 38aa ENSMUSP00000157508.1 Protein coding - A0A494B969 CDS 3' 204 incomplete

Add3- ENSMUST00000237593.1 380 71aa ENSMUSP00000158538.1 Protein coding - A0A494BBK1 CDS 3' 214 incomplete

Add3- ENSMUST00000237153.1 95 3aa ENSMUSP00000158206.1 Protein coding - - CDS 3' 210 incomplete

Add3- ENSMUST00000236417.1 1278 No - Retained - - - 208 protein intron

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126.96 kb Forward strand 53.14Mb 53.16Mb 53.18Mb 53.20Mb 53.22Mb 53.24Mb Genes (Comprehensive set... Gm30381-201 >lncRNA Add3-206 >protein coding

Add3-203 >protein coding

Add3-205 >protein coding

Add3-208 >retained intron

Add3-212 >protein coding

Add3-216 >protein coding

Add3-204 >protein coding

Add3-211 >protein coding

Add3-202 >protein coding

Add3-215 >protein coding

Add3-213 >protein coding

Add3-214 >protein coding

Add3-201 >protein coding

Add3-207 >protein coding

Add3-209 >protein coding

Add3-210 >protein coding

Contigs < AC138174.9 Genes < Gm9618-201processed pseudogen

< 1700001K23Rik-205lncRNA

< 1700001K23Rik-202lncRNA

< 1700001K23Rik-201lncRNA

< 1700001K23Rik-204lncRNA

Regulatory Build

53.14Mb 53.16Mb 53.18Mb 53.20Mb 53.22Mb 53.24Mb Reverse strand 126.96 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

pseudogene RNA gene processed transcript

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Transcript: ENSMUST00000237301

106.44 kb Forward strand

Add3-212 >protein coding

ENSMUSP00000158... 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 Gamma-adducin

PTHR10672 Gene3D Class II aldolase/adducin N-terminal domain superfamily

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 706

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

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