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Mouse Akap7 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Akap7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Akap7 gene (NCBI Reference Sequence: NM_018747 ; Ensembl: ENSMUSG00000039166 ) is located on Mouse chromosome 10. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000041984). 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 Akap7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-234K7 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 are viable and fertile and show normal cardiomyocyte response to adrenergic stimulation.

Exon 3 starts from about 4.35% 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: 5557 bp, and the size of intron 3 for 3'-loxP site insertion: 12251 bp. The size of effective cKO region: ~649 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 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Akap7 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(7149bp) | A(28.07% 2007) | C(19.15% 1369) | T(31.04% 2219) | G(21.74% 1554)

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% chr10 - 25284292 25287291 3000 browser details YourSeq 171 2256 2442 3000 96.3% chr3 - 127457844 127458039 196 browser details YourSeq 169 2256 2442 3000 95.8% chr1 + 181036970 181037171 202 browser details YourSeq 167 2256 2442 3000 95.2% chr11 + 115391113 115391326 214 browser details YourSeq 165 2256 2447 3000 93.7% chr16 - 4961537 5266160 304624 browser details YourSeq 165 2250 2444 3000 91.4% chr9 + 88586254 88586443 190 browser details YourSeq 163 2256 2449 3000 90.1% chr17 - 66271219 66271409 191 browser details YourSeq 163 2256 2442 3000 94.1% chr2 + 90888761 90973857 85097 browser details YourSeq 161 2247 2442 3000 93.1% chr3 - 97028469 97028731 263 browser details YourSeq 161 2256 2442 3000 94.5% chr19 - 5396881 5397068 188 browser details YourSeq 160 2256 2448 3000 92.7% chr18 + 6807186 6807383 198 browser details YourSeq 160 2257 2448 3000 90.4% chr15 + 103465937 103466125 189 browser details YourSeq 159 2048 2442 3000 80.2% chr4 + 41421621 41421865 245 browser details YourSeq 159 2256 2446 3000 92.6% chr11 + 35900775 35900973 199 browser details YourSeq 158 2258 2442 3000 91.3% chrX - 151636312 151636494 183 browser details YourSeq 158 2219 2442 3000 91.2% chr9 + 59646622 59646860 239 browser details YourSeq 157 2259 2447 3000 90.2% chr6 - 88438636 88438821 186 browser details YourSeq 157 2241 2438 3000 91.6% chr4 - 141701661 141701858 198 browser details YourSeq 157 2257 2447 3000 90.4% chr2 - 14993336 14993524 189 browser details YourSeq 157 2257 2442 3000 92.5% chr10 - 76064295 76064485 191

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% chr10 - 25280643 25283642 3000 browser details YourSeq 127 944 1137 3000 87.5% chr9 - 88706002 88706201 200 browser details YourSeq 125 970 1149 3000 88.4% chr3 - 86538547 86691375 152829 browser details YourSeq 121 995 1149 3000 89.5% chr11 - 112677156 112677316 161 browser details YourSeq 118 970 1165 3000 81.3% chr6 - 37405434 37405701 268 browser details YourSeq 116 974 1149 3000 86.8% chr18 - 3648629 3648808 180 browser details YourSeq 114 1001 1149 3000 90.2% chr8 + 77130326 77130480 155 browser details YourSeq 112 995 1148 3000 90.6% chr11 + 53002750 53002908 159 browser details YourSeq 110 998 1149 3000 89.3% chr4 - 53688817 53688973 157 browser details YourSeq 110 1009 1149 3000 89.3% chr18 - 88797482 88797628 147 browser details YourSeq 110 995 1285 3000 93.0% chr12 - 109852154 109852480 327 browser details YourSeq 109 990 1149 3000 85.8% chr9 - 52931952 52932118 167 browser details YourSeq 109 996 1150 3000 89.3% chr11 - 70260537 70260697 161 browser details YourSeq 109 968 1126 3000 88.2% chr10 + 4020785 4020947 163 browser details YourSeq 107 1008 1149 3000 88.1% chr7 - 137539745 137539892 148 browser details YourSeq 107 974 1133 3000 92.8% chr10 - 45708861 45709032 172 browser details YourSeq 107 995 1138 3000 89.1% chr9 + 18739646 18739792 147 browser details YourSeq 107 964 1149 3000 85.9% chr13 + 116906136 116906327 192 browser details YourSeq 106 1004 1150 3000 89.7% chr18 - 78049687 78049837 151 browser details YourSeq 105 995 1135 3000 91.2% chr17 - 43881490 43881632 143

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: Akap7 A kinase (PRKA) anchor protein 7 [ Mus musculus (house mouse) ] Gene ID: 432442, updated on 12-Aug-2019

Gene summary

Official Symbol Akap7 provided by MGI Official Full Name A kinase (PRKA) anchor protein 7 provided by MGI Primary source MGI:MGI:1859150 See related Ensembl:ENSMUSG00000039166 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 AKAP15; Akap18; AI662165; BB170514; 6430401D08 Expression Broad expression in cerebellum adult (RPKM 6.0), CNS E18 (RPKM 4.9) and 24 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 A4 See Akap7 in Genome Data Viewer

Exon count: 19

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (25169090..25299163, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (24888896..25018969, complement)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 8 transcripts

Gene: Akap7 ENSMUSG00000039166

Description A kinase (PRKA) anchor protein 7 [Source:MGI Symbol;Acc:MGI:1859150] Gene Synonyms AKAP15, Akap18 Location Chromosome 10: 25,169,090-25,307,870 reverse strand. GRCm38:CM001003.2 About this gene This gene has 8 transcripts (splice variants), 189 orthologues, 1 paralogue, is a member of 2 Ensembl protein families and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Akap7-203 ENSMUST00000100012.2 2321 81aa ENSMUSP00000097590.2 Protein coding CCDS83688 O55074 TSL:1 GENCODE basic

Akap7-201 ENSMUST00000041984.13 2092 314aa ENSMUSP00000043624.7 Protein coding CCDS23753 Q7TN79 TSL:1 GENCODE basic APPRIS P1

Akap7-202 ENSMUST00000095779.10 919 122aa ENSMUSP00000093455.4 Protein coding - Q4KL21 TSL:1 GENCODE basic

Akap7-206 ENSMUST00000176258.7 720 141aa ENSMUSP00000135037.1 Protein coding - H3BJL8 CDS 3' incomplete TSL:3

Akap7-208 ENSMUST00000220347.1 557 95aa ENSMUSP00000151597.1 Protein coding - A0A1W2P7B3 CDS 5' incomplete TSL:2

Akap7-207 ENSMUST00000177124.1 397 126aa ENSMUSP00000135137.1 Protein coding - H3BJV2 CDS 3' incomplete TSL:2

Akap7-205 ENSMUST00000176231.1 236 19aa ENSMUSP00000135433.1 Protein coding - H3BKK9 CDS 3' incomplete TSL:5

Akap7-204 ENSMUST00000175979.1 2391 No protein - lncRNA - - TSL:1

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158.78 kb Forward strand 25.20Mb 25.25Mb 25.30Mb Genes Gm7791-201 >processed pseudogene Gm40617-201 >lncRNA (Comprehensive set...

Contigs < AC153549.5 < AC153550.10 Genes (Comprehensive set... < Akap7-204lncRNA < Gm25016-201snoRNA

< Akap7-203protein coding < Akap7-206protein coding

< Akap7-208protein coding < Akap7-202protein coding

< Akap7-201protein coding

< Gm26167-201misc RNA

< Akap7-207protein coding

< Akap7-205protein coding

Regulatory Build

25.20Mb 25.25Mb 25.30Mb Reverse strand 158.78 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 pseudogene

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Transcript: ENSMUST00000041984

< Akap7-201protein coding

Reverse strand 129.04 kb

ENSMUSP00000043... MobiDB lite Low complexity (Seg) Superfamily Cyclic phosphodiesterase

Pfam Protein kinase A anchor protein, nuclear localisation signal domain A-kinase anchor protein 7, RI-RII subunit-binding domain

PANTHER PTHR15934

PTHR15934:SF6 Gene3D 3.90.1140.10

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 314

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

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