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Mouse Akna Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Akna gene (NCBI Reference Sequence: NM_001045514 ; Ensembl: ENSMUSG00000039158 ) is located on Mouse chromosome 4. 22 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 22 (Transcript: ENSMUST00000035724). 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 Akna gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-322B16 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 hypomorphic or a knock-out allele exhibit partial postnatal lethality, pathogen-induced acute neutrophil responses leading to systemic inflammation and alveolar destruction, and increased susceptibility to fungal infection.

Exon 3 starts from about 5.98% 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: 2240 bp, and the size of intron 3 for 3'-loxP site insertion: 2379 bp. The size of effective cKO region: ~1549 bp. The cKO 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 2 3 22 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Akna 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8049bp) | A(22.65% 1823) | C(26.24% 2112) | T(25.0% 2012) | G(26.12% 2102)

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% chr4 - 63395885 63398884 3000 browser details YourSeq 99 2420 2566 3000 90.1% chr15 - 84327196 84327414 219 browser details YourSeq 88 2420 2652 3000 77.2% chr14 - 58138831 58139000 170 browser details YourSeq 86 2406 2565 3000 78.3% chr10 - 84463399 84463526 128 browser details YourSeq 81 2426 2565 3000 88.4% chr10 + 79641052 79641365 314 browser details YourSeq 80 2425 2563 3000 89.6% chr12 - 25478894 25479112 219 browser details YourSeq 76 2461 2585 3000 88.0% chr13 - 103756594 103756743 150 browser details YourSeq 75 2406 2648 3000 89.4% chr12 - 102383872 102384152 281 browser details YourSeq 70 2405 2565 3000 82.0% chr12 + 109142010 109142157 148 browser details YourSeq 69 2457 2565 3000 86.8% chr15 + 28039325 28039430 106 browser details YourSeq 68 2454 2565 3000 88.7% chr11 - 90455565 90455696 132 browser details YourSeq 68 2462 2568 3000 91.5% chr12 + 109062688 109062907 220 browser details YourSeq 67 2462 2587 3000 76.9% chr1 - 56801987 56802081 95 browser details YourSeq 67 2473 2644 3000 74.1% chr11 + 19398379 19398500 122 browser details YourSeq 66 2461 2565 3000 88.3% chr1 - 164539845 164540062 218 browser details YourSeq 64 2473 2565 3000 92.0% chr11 - 90315919 90316103 185 browser details YourSeq 64 2461 2565 3000 78.1% chr15 + 87581811 87581884 74 browser details YourSeq 63 2457 2565 3000 89.5% chr14 - 60668070 60668287 218 browser details YourSeq 56 2448 2565 3000 91.2% chr14 - 48306562 48306831 270 browser details YourSeq 55 2495 2568 3000 89.8% chr2 + 173396571 173396661 91

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% chr4 - 63391336 63394335 3000 browser details YourSeq 110 985 1515 3000 91.8% chr15 - 84651971 84652803 833 browser details YourSeq 91 1018 1516 3000 74.8% chr1 - 86438439 86438779 341 browser details YourSeq 80 995 1223 3000 67.7% chrX - 10771483 10771712 230 browser details YourSeq 79 997 1223 3000 82.3% chr1 - 127867725 127867924 200 browser details YourSeq 77 1002 1221 3000 89.7% chr4 - 119186211 119186431 221 browser details YourSeq 76 1431 1528 3000 92.4% chr2 - 158455595 158650575 194981 browser details YourSeq 76 1425 1534 3000 84.6% chr1 - 66882958 66883067 110 browser details YourSeq 75 1385 1528 3000 85.1% chr2 - 166823308 166823442 135 browser details YourSeq 72 1035 1223 3000 89.2% chr7 - 134905299 134905487 189 browser details YourSeq 71 990 1220 3000 91.9% chr6 - 88217565 88218079 515 browser details YourSeq 71 1015 1221 3000 81.7% chr12 - 111174428 111174622 195 browser details YourSeq 71 981 1516 3000 72.7% chr1 + 6196961 6197252 292 browser details YourSeq 69 1000 1222 3000 92.6% chr1 + 86642191 86642603 413 browser details YourSeq 68 1384 1521 3000 78.8% chr7 + 126216399 126216534 136 browser details YourSeq 68 1422 1525 3000 82.7% chr6 + 146492076 146492179 104 browser details YourSeq 65 1424 1528 3000 89.4% chr2 - 173726716 173726818 103 browser details YourSeq 64 1429 1520 3000 90.0% chr1 - 192118355 192118447 93 browser details YourSeq 63 1002 1220 3000 93.2% chr4 - 86902752 86903645 894 browser details YourSeq 63 1384 1521 3000 86.5% chr1 - 36677930 36678066 137

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: Akna AT-hook transcription factor [ Mus musculus (house mouse) ] Gene ID: 100182, updated on 12-Aug-2019

Gene summary

Official Symbol Akna provided by MGI Official Full Name AT-hook transcription factor provided by MGI Primary source MGI:MGI:2140340 See related Ensembl:ENSMUSG00000039158 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 AI597013 Expression Biased expression in spleen adult (RPKM 37.5), thymus adult (RPKM 21.8) and 13 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 B3-C1 See Akna in Genome Data Viewer

Exon count: 26

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (63367123..63411094, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (63028161..63064479, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 2 transcripts

Gene: Akna ENSMUSG00000039158

Description AT-hook transcription factor [Source:MGI Symbol;Acc:MGI:2140340] Location Chromosome 4: 63,367,125-63,403,354 reverse strand. GRCm38:CM000997.2 About this gene This gene has 2 transcripts (splice variants), 151 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Akna-201 ENSMUST00000035724.4 5387 1404aa ENSMUSP00000041614.4 Protein coding CCDS38777 Q80VW7 TSL:5 GENCODE basic APPRIS P1

Akna-202 ENSMUST00000140586.1 707 No protein - lncRNA - - TSL:3

56.23 kb Forward strand 63.36Mb 63.37Mb 63.38Mb 63.39Mb 63.40Mb 63.41Mb Genes Orm3-201 >protein coding Aknaos-201 >lncRNA (Comprehensive set...

Orm2-201 >protein coding

Contigs AL683828.8 >

Genes (Comprehensive set... < Akna-201protein coding

< Akna-202lncRNA

Regulatory Build

63.36Mb 63.37Mb 63.38Mb 63.39Mb 63.40Mb 63.41Mb Reverse strand 56.23 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000035724

< Akna-201protein coding

Reverse strand 36.23 kb

ENSMUSP00000041... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Transcription factor, AT-hook-containing PANTHER Transcription factor, AT-hook-containing

PTHR21510:SF15

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

Variant Legend inframe insertion inframe deletion missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1404

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

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