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Mouse Azi2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Azi2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Azi2 (NCBI Reference Sequence: NM_013727 ; Ensembl: ENSMUSG00000039285 ) is located on Mouse 9. 8 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 8 (Transcript: ENSMUST00000044454). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Azi2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-243F19 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 a knock-out allele exhibit impaired GM-CSF-derived bone marrow-derived dendritic cell differenatiation, cytokine response and ability to stimulate T cells.

Exon 6 starts from about 48.72% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 4313 bp, and the size of intron 6 for 3'-loxP site insertion: 3146 bp. The size of effective cKO region: ~595 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 6 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Azi2 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(7095bp) | A(32.47% 2304) | C(16.84% 1195) | T(30.36% 2154) | G(20.32% 1442)

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% chr9 + 118052567 118055566 3000 browser details YourSeq 44 272 331 3000 94.0% chr13 + 31150012 31150114 103 browser details YourSeq 42 2079 2149 3000 95.7% chr5 - 118021241 118021313 73 browser details YourSeq 40 2089 2148 3000 93.7% chr17 + 29725810 29725871 62 browser details YourSeq 39 2078 2149 3000 95.4% chr4 - 109825804 109825876 73 browser details YourSeq 38 292 331 3000 97.5% chr7 - 91594522 91594561 40 browser details YourSeq 38 294 333 3000 97.5% chr12 - 76829584 76829623 40 browser details YourSeq 38 283 327 3000 93.1% chr10 - 105564622 105564672 51 browser details YourSeq 37 2064 2102 3000 92.2% chr10 - 128174833 128174870 38 browser details YourSeq 37 293 331 3000 97.5% chr1 - 122513460 122513498 39 browser details YourSeq 36 292 339 3000 89.2% chr1 - 7911678 7911726 49 browser details YourSeq 34 293 331 3000 97.3% chrX + 11578179 11578217 39 browser details YourSeq 34 294 328 3000 100.0% chr2 + 172224400 172224450 51 browser details YourSeq 33 292 331 3000 80.6% chrX - 116668990 116669025 36 browser details YourSeq 33 294 328 3000 97.2% chr2 + 9533515 9533549 35 browser details YourSeq 33 292 331 3000 80.6% chr19 + 15351678 15351713 36 browser details YourSeq 33 2086 2149 3000 94.6% chr14 + 73670978 73671074 97 browser details YourSeq 33 299 331 3000 100.0% chr12 + 65980036 65980068 33 browser details YourSeq 33 292 328 3000 94.6% chr11 + 54456621 54456657 37 browser details YourSeq 32 292 323 3000 100.0% chr2 - 79063329 79063360 32

Note: The 3000 bp section upstream of Exon 6 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% chr9 + 118056162 118059161 3000 browser details YourSeq 149 394 739 3000 84.1% chr17 + 14880831 14881018 188 browser details YourSeq 146 384 557 3000 94.2% chr16 - 88395575 88395757 183 browser details YourSeq 146 384 717 3000 82.4% chr9 + 64279195 64279386 192 browser details YourSeq 144 394 717 3000 85.0% chr11 - 96832102 96832288 187 browser details YourSeq 144 384 555 3000 94.0% chr12 + 108498831 108499011 181 browser details YourSeq 144 384 557 3000 93.0% chr11 + 79946655 79946842 188 browser details YourSeq 143 394 720 3000 85.5% chr3 - 55872041 55872224 184 browser details YourSeq 143 384 557 3000 93.0% chr6 + 72408574 72408757 184 browser details YourSeq 143 365 555 3000 93.5% chr11 + 86547495 86548052 558 browser details YourSeq 142 384 558 3000 92.4% chr11 - 51717827 51718013 187 browser details YourSeq 142 384 557 3000 93.0% chr10 - 81829011 81829236 226 browser details YourSeq 142 384 556 3000 93.0% chr3 + 131262093 131262275 183 browser details YourSeq 141 384 588 3000 90.4% chr4 - 146487169 146487379 211 browser details YourSeq 141 384 557 3000 92.8% chr5 + 22030314 22030496 183 browser details YourSeq 141 384 557 3000 92.8% chr2 + 92040615 92040798 184 browser details YourSeq 140 384 554 3000 92.4% chr19 + 53213373 53213558 186 browser details YourSeq 139 384 557 3000 92.8% chr8 - 122606069 122606255 187 browser details YourSeq 139 384 557 3000 92.3% chr4 - 140359261 140359443 183 browser details YourSeq 139 385 546 3000 94.4% chr10 - 13523058 13523267 210

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Azi2 5-azacytidine induced gene 2 [ Mus musculus (house mouse) ] Gene ID: 27215, updated on 12-Aug-2019

Gene summary

Official Symbol Azi2 provided by MGI Official Full Name 5-azacytidine induced gene 2 provided by MGI Primary source MGI:MGI:1351332 See related Ensembl:ENSMUSG00000039285 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 AZ2; AA410145 Expression Ubiquitous expression in adrenal adult (RPKM 8.1), bladder adult (RPKM 7.9) and 28 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 F3 See Azi2 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (118040442..118069798)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (117949617..117973025)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 10 transcripts

Gene: Azi2 ENSMUSG00000039285

Description 5-azacytidine induced gene 2 [Source:MGI Symbol;Acc:MGI:1351332] Gene Synonyms AZ2 Location Chromosome 9: 118,040,499-118,069,794 forward strand. GRCm38:CM001002.2 About this gene This gene has 10 transcripts (splice variants), 206 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Azi2- ENSMUST00000044454.11 3396 405aa ENSMUSP00000044350.5 Protein coding CCDS40799 Q9QYP6 TSL:1 201 GENCODE basic APPRIS P1

Azi2- ENSMUST00000134433.7 2499 283aa ENSMUSP00000114980.1 Protein coding CCDS52959 Q9QYP6 TSL:1 207 GENCODE basic

Azi2- ENSMUST00000133580.7 2156 405aa ENSMUSP00000118765.1 Protein coding CCDS40799 Q9QYP6 TSL:1 205 GENCODE basic APPRIS P1

Azi2- ENSMUST00000130735.7 1407 199aa ENSMUSP00000114634.1 Protein coding - F7A092 CDS 5' 204 incomplete TSL:3

Azi2- ENSMUST00000135251.1 742 178aa ENSMUSP00000116971.1 Protein coding - D3Z7S3 CDS 3' 208 incomplete TSL:2

Azi2- ENSMUST00000123690.1 735 67aa ENSMUSP00000121245.1 Protein coding - F6SY96 CDS 5' 202 incomplete TSL:5

Azi2- ENSMUST00000154583.7 1817 114aa ENSMUSP00000122063.1 Nonsense mediated - Q9QYP6 TSL:1 210 decay

Azi2- ENSMUST00000143024.1 943 No - Retained intron - - TSL:2 209 protein

Azi2- ENSMUST00000133814.1 599 No - Retained intron - - TSL:2 206 protein

Azi2- ENSMUST00000127189.1 466 No - Retained intron - - TSL:2 203 protein

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49.30 kb Forward strand

118.04Mb 118.05Mb 118.06Mb 118.07Mb Azi2-201 >protein coding (Comprehensive set...

Azi2-205 >protein coding

Azi2-207 >protein coding

Azi2-210 >nonsense mediated decay

Azi2-209 >retained intron Azi2-208 >protein coding

Azi2-204 >protein coding

Azi2-203 >retained intron Azi2-202 >protein coding

Azi2-206 >retained intron

Contigs AC157475.2 >

Genes < Zcwpw2-202retained intron < Cmc1-201protein coding (Comprehensive set...

< Cmc1-202protein coding

Regulatory Build

118.04Mb 118.05Mb 118.06Mb 118.07Mb Reverse strand 49.30 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

processed transcript

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Transcript: ENSMUST00000044454

23.34 kb Forward strand

Azi2-201 >protein coding

ENSMUSP00000044... Coiled-coils (Ncoils) Pfam Tbk1/Ikki binding domain PANTHER PTHR14432:SF6

PTHR14432

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 405

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

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