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Mouse Akap4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Akap4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Akap4 gene (NCBI Reference Sequence: NM_009651 ; Ensembl: ENSMUSG00000050089 ) is located on Mouse chromosome X. 6 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000057101). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Akap4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-372F22 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: Inactivation of this locus results in male infertility due to impaired sperm motility. Heterozygous mutant females show no reproductive defects.

Exon 5 is not frameshift exon, and covers 83.16% of the coding region. The size of intron 4 for 5'-loxP site insertion: 1972 bp, and the size of intron 5 for 3'-loxP site insertion: 437 bp. The size of effective cKO region: ~2586 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 4 5 6 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Akap4 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(9054bp) | A(31.86% 2885) | C(21.06% 1907) | T(25.49% 2308) | G(21.58% 1954)

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% chrX + 7072460 7075459 3000 browser details YourSeq 160 1 267 3000 91.8% chrX + 7071232 7072164 933 browser details YourSeq 110 25 254 3000 90.3% chrX + 7071655 7072076 422 browser details YourSeq 93 25 258 3000 91.0% chrX + 7071003 7071321 319 browser details YourSeq 70 24 114 3000 85.8% chrX + 7071485 7071570 86 browser details YourSeq 59 2074 2633 3000 61.4% chr1 + 34414616 34414783 168 browser details YourSeq 54 2133 2221 3000 83.0% chr11 + 62829436 62829537 102 browser details YourSeq 52 2076 2179 3000 95.0% chr5 + 122364389 122364512 124 browser details YourSeq 51 2074 2179 3000 76.9% chr11 + 80068830 80069158 329 browser details YourSeq 50 2060 2177 3000 93.2% chr9 + 56921242 56921363 122 browser details YourSeq 49 2075 2157 3000 82.1% chr16 - 17035229 17035311 83 browser details YourSeq 49 2073 2159 3000 78.2% chr11 + 68237419 68237505 87 browser details YourSeq 47 2141 2260 3000 89.9% chr7 + 16324230 16324349 120 browser details YourSeq 46 2074 2159 3000 76.8% chr19 - 4563187 4563272 86 browser details YourSeq 45 2077 2159 3000 77.2% chr7 - 101910939 101911021 83 browser details YourSeq 45 2063 2157 3000 70.0% chr5 - 99502493 99502584 92 browser details YourSeq 45 2077 2159 3000 77.2% chr1 - 43932323 43932405 83 browser details YourSeq 45 2164 2502 3000 64.0% chr3 + 29718753 29718851 99 browser details YourSeq 45 2077 2216 3000 83.6% chr11 + 67834904 67835051 148 browser details YourSeq 44 2073 2171 3000 94.0% chr18 - 12218658 12218768 111

Note: The 3000 bp section upstream of Exon 5 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% chrX + 7078046 7081045 3000 browser details YourSeq 101 831 991 3000 92.7% chr8 + 87898007 87898175 169 browser details YourSeq 89 869 991 3000 94.3% chr8 + 61705351 61705500 150 browser details YourSeq 88 831 991 3000 84.8% chr8 - 9806797 9806944 148 browser details YourSeq 83 829 960 3000 93.7% chr17 - 90410199 90410371 173 browser details YourSeq 82 833 971 3000 83.6% chr13 - 109894361 109894482 122 browser details YourSeq 65 834 978 3000 76.0% chr5 + 119600004 119600088 85 browser details YourSeq 64 832 928 3000 93.5% chr4 - 154065659 154065770 112 browser details YourSeq 63 835 920 3000 94.6% chr7 - 15959564 15959716 153 browser details YourSeq 62 831 928 3000 93.3% chr4 + 152443694 152443826 133 browser details YourSeq 61 880 990 3000 80.0% chr7 - 16726251 16726334 84 browser details YourSeq 60 831 918 3000 83.9% chr17 - 90410215 90410291 77 browser details YourSeq 55 882 967 3000 78.2% chrY - 81337306 81337374 69 browser details YourSeq 55 912 990 3000 96.8% chr10 - 73003335 73003549 215 browser details YourSeq 53 879 968 3000 79.4% chr10 - 90592811 90592884 74 browser details YourSeq 51 835 901 3000 91.0% chr7 - 91884195 91884259 65 browser details YourSeq 51 831 881 3000 100.0% chr10 - 124250626 124250676 51 browser details YourSeq 47 878 968 3000 94.3% chr2 + 30610256 30610543 288 browser details YourSeq 43 831 893 3000 96.0% chr13 + 98649728 98649802 75 browser details YourSeq 39 948 988 3000 97.6% chr13 + 98649608 98649648 41

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Akap4 A kinase (PRKA) anchor protein 4 [ Mus musculus (house mouse) ] Gene ID: 11643, updated on 12-Aug-2019

Gene summary

Official Symbol Akap4 provided by MGI Official Full Name A kinase (PRKA) anchor protein 4 provided by MGI Primary source MGI:MGI:102794 See related Ensembl:ENSMUSG00000050089 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 p82; Fsc1; PRKA4; AKAP-4; AKAP82; mAKAP82 Expression Restricted expression toward testis adult (RPKM 214.6) See more Orthologs human all

Genomic context

Location: X A1.1; X 3.17 cM See Akap4 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (7067515..7078609)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (6644645..6655735)

Chromosome X - NC_000086.7

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Transcript information: This gene has 3 transcripts

Gene: Akap4 ENSMUSG00000050089

Description A kinase (PRKA) anchor protein 4 [Source:MGI Symbol;Acc:MGI:102794] Gene Synonyms Fsc1 Location Chromosome X: 7,067,519-7,078,609 forward strand. GRCm38:CM001013.2 About this gene This gene has 3 transcripts (splice variants), 114 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Akap4-202 ENSMUST00000115750.7 3021 840aa ENSMUSP00000111416.1 Protein coding CCDS40839 Q60662 TSL:5 GENCODE basic APPRIS ALT2

Akap4-201 ENSMUST00000057101.12 2851 849aa ENSMUSP00000050962.6 Protein coding CCDS40838 A1L3T6 Q60662 TSL:1 GENCODE basic APPRIS P4

Akap4-203 ENSMUST00000115751.2 2838 840aa ENSMUSP00000111417.1 Protein coding CCDS40839 Q60662 TSL:1 GENCODE basic APPRIS ALT2

31.09 kb Forward strand 7.06Mb 7.07Mb 7.08Mb Genes (Comprehensive set... Akap4-201 >protein coding

Akap4-202 >protein coding

Akap4-203 >protein coding

Contigs AL808124.12 > Regulatory Build

7.06Mb 7.07Mb 7.08Mb Reverse strand 31.09 kb

Regulation Legend

Enhancer

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

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Transcript: ENSMUST00000057101

11.09 kb Forward strand

Akap4-201 >protein coding

ENSMUSP00000050... MobiDB lite Low complexity (Seg) SMART A-kinase anchor 110kDa Pfam RII binding domain

A-kinase anchor 110kDa, C-terminal PANTHER PTHR10226:SF8

SPHK1-interactor/A-kinase anchor 110kDa

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 849

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

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