https://www.alphaknockout.com

Mouse Aig1 Knockout Project (CRISPR/Cas9)

Objective: To create a Aig1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Aig1 (NCBI Reference Sequence: NM_025446 ; Ensembl: ENSMUSG00000019806 ) is located on Mouse 10. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000019942). Exon 2 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:

Exon 2 starts from about 18.07% of the coding region. Exon 2 covers 19.85% of the coding region. The size of effective KO region: ~156 bp. The KO 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 2 6

Legends Exon of mouse Aig1 Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 156 bp section 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 156 bp section 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.

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

Sequence 12

Summary: Full Length(156bp) | A(16.67% 26) | C(29.49% 46) | T(23.72% 37) | G(30.13% 47)

Note: The 156 bp section 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(156bp) | A(16.67% 26) | C(29.49% 46) | T(23.08% 36) | G(30.77% 48)

Note: The 156 bp section 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 156 1 156 156 100.0% chr10 - 13829242 13829397 156 browser details YourSeq 23 23 48 156 96.2% chr1 + 35982676 35982712 37 browser details YourSeq 22 90 112 156 100.0% chr11 + 47121048 47121072 25 browser details YourSeq 21 94 114 156 100.0% chr17 - 65420543 65420563 21

Note: The 156 bp section 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 156 1 156 156 100.0% chr10 - 13829244 13829399 156 browser details YourSeq 22 92 114 156 100.0% chr11 + 47121048 47121072 25 browser details YourSeq 21 96 116 156 100.0% chr17 - 65420543 65420563 21

Note: The 156 bp section of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Aig1 androgen-induced 1 [ Mus musculus (house mouse) ] Gene ID: 66253, updated on 10-Oct-2019

Gene summary

Official Symbol Aig1 provided by MGI Official Full Name androgen-induced 1 provided by MGI Primary source MGI:MGI:1913503 See related Ensembl:ENSMUSG00000019806 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 CGI-103; AV064870; AW413422; 1500031O19Rik Expression Ubiquitous expression in cerebellum adult (RPKM 19.7), colon adult (RPKM 16.9) and 27 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 A2 See Aig1 in Genome Data Viewer Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (13647054..13869038, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (13372515..13588636, complement)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 7 transcripts

Gene: Aig1 ENSMUSG00000019806

Description androgen-induced 1 [Source:MGI Symbol;Acc:MGI:1913503] Gene Synonyms 1500031O19Rik, CGI-103 Location Chromosome 10: 13,647,054-13,868,980 reverse strand. GRCm38:CM001003.2 About this gene This gene has 7 transcripts (splice variants), 199 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Aig1-205 ENSMUST00000162610.7 1554 238aa ENSMUSP00000125366.1 Protein coding CCDS83682 Q3TS32 Q9D8B1 TSL:1 GENCODE basic APPRIS P1

Aig1-201 ENSMUST00000019942.5 1439 262aa ENSMUSP00000019942.5 Protein coding CCDS23703 Q9D8B1 TSL:1 GENCODE basic

Aig1-202 ENSMUST00000105534.9 617 180aa ENSMUSP00000101174.3 Protein coding - H7BX91 TSL:5 GENCODE basic

Aig1-206 ENSMUST00000162798.1 4369 No protein - Retained intron - - TSL:1

Aig1-203 ENSMUST00000161729.1 3254 No protein - Retained intron - - TSL:1

Aig1-204 ENSMUST00000162174.7 642 No protein - lncRNA - - TSL:3

Aig1-207 ENSMUST00000162869.1 441 No protein - lncRNA - - TSL:5

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241.93 kb Forward strand 13.65Mb 13.70Mb 13.75Mb 13.80Mb 13.85Mb Gm32172-201 >lncRNA (Comprehensive set...

Contigs AC160027.16 > < AC134867.3 Genes (Comprehensive set... < Aig1-205protein coding

< Aig1-202protein coding

< Aig1-201protein coding

< Aig1-204lncRNA < Gm32105-201lncRNA

< Aig1-206retained intron

< Aig1-203retained intron

< Aig1-207lncRNA

Regulatory Build

13.65Mb 13.70Mb 13.75Mb 13.80Mb 13.85Mb Reverse strand 241.93 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: ENSMUST00000019942

< Aig1-201protein coding

Reverse strand 216.51 kb

ENSMUSP00000019... Transmembrane heli... Low complexity (Seg) Pfam FAR-17a/AIG1-like protein PANTHER FAR-17a/AIG1-like protein

PTHR10989:SF11

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 262

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

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