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Mouse Asic5 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Asic5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Asic5 gene (NCBI Reference Sequence: NM_021370 ; Ensembl: ENSMUSG00000028008 ) is located on Mouse chromosome 3. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000029641). 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 Asic5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-353B19 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:

Exon 3 starts from about 23.43% 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: 4555 bp, and the size of intron 3 for 3'-loxP site insertion: 1796 bp. The size of effective cKO region: ~738 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 4 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Asic5 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(7238bp) | A(33.31% 2411) | C(17.6% 1274) | T(30.46% 2205) | G(18.62% 1348)

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% chr3 + 82001197 82004196 3000 browser details YourSeq 49 1273 1370 3000 93.0% chr15 + 85649710 85650071 362 browser details YourSeq 42 1258 1326 3000 73.4% chr10 - 22272156 22272216 61 browser details YourSeq 39 1272 1324 3000 89.8% chr10 + 86852548 86852622 75 browser details YourSeq 38 1250 1324 3000 81.7% chr11 - 113234940 113235049 110 browser details YourSeq 38 1265 1324 3000 75.0% chr11 + 4468178 4468227 50 browser details YourSeq 38 1273 1324 3000 95.5% chr10 + 35948336 35948501 166 browser details YourSeq 38 1259 1319 3000 93.4% chr10 + 22904741 22904804 64 browser details YourSeq 36 1255 1323 3000 76.2% chr8 - 8167189 8167249 61 browser details YourSeq 36 1258 1326 3000 95.0% chr17 - 57476725 57476933 209 browser details YourSeq 36 1253 1312 3000 75.0% chr11 - 55630779 55630826 48 browser details YourSeq 36 1272 1326 3000 91.0% chr12 + 37081642 37081716 75 browser details YourSeq 36 1281 1326 3000 91.0% chr1 + 90956221 90956286 66 browser details YourSeq 35 1273 1325 3000 92.2% chr11 - 3152986 3153037 52 browser details YourSeq 33 1310 1350 3000 89.2% chr11 + 56015376 56015415 40 browser details YourSeq 32 1273 1319 3000 94.5% chr5 + 112926008 112926056 49 browser details YourSeq 32 1273 1325 3000 86.2% chr17 + 50219766 50219816 51 browser details YourSeq 31 2649 2693 3000 84.5% chrX + 117114957 117115001 45 browser details YourSeq 30 1896 1931 3000 80.7% chr17 - 38577387 38577417 31 browser details YourSeq 29 1281 1326 3000 69.7% chr15 - 44796475 44796508 34

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% chr3 + 82004935 82007934 3000 browser details YourSeq 72 2916 3000 3000 93.0% chr9 - 8096937 8097022 86 browser details YourSeq 72 2915 3000 3000 89.5% chr10 - 121025277 121025361 85 browser details YourSeq 71 2900 3000 3000 92.8% chrX - 53065452 53065840 389 browser details YourSeq 71 2900 3000 3000 80.9% chrX + 109501540 109501633 94 browser details YourSeq 70 2916 2995 3000 93.8% chr7 - 4800981 4801060 80 browser details YourSeq 69 2907 3000 3000 82.3% chr4 + 33006461 33006550 90 browser details YourSeq 68 2894 3000 3000 77.6% chr5 - 33302048 33302136 89 browser details YourSeq 68 2922 3000 3000 93.7% chr10 - 69597197 69973509 376313 browser details YourSeq 68 2897 3000 3000 81.2% chr2 + 102353551 102353648 98 browser details YourSeq 67 2922 2998 3000 93.6% chr7 - 43817977 43818053 77 browser details YourSeq 67 2922 3000 3000 92.5% chr10 + 126953833 126953911 79 browser details YourSeq 67 2922 3000 3000 92.5% chr1 + 119808347 119808425 79 browser details YourSeq 65 2926 3000 3000 93.4% chr8 - 111216098 111216172 75 browser details YourSeq 65 2926 3000 3000 93.4% chr7 - 87786606 87786680 75 browser details YourSeq 65 2898 3000 3000 84.3% chr5 - 83241603 83241725 123 browser details YourSeq 65 2902 3000 3000 84.3% chr17 - 29672335 29672435 101 browser details YourSeq 64 2921 3000 3000 90.0% chr19 - 42896563 42896642 80 browser details YourSeq 64 2929 3000 3000 94.5% chr10 + 70874886 70874957 72 browser details YourSeq 63 2918 3000 3000 88.0% chr6 - 117847724 117847806 83

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: Asic5 acid-sensing (proton-gated) ion channel family member 5 [ Mus musculus (house mouse) ] Gene ID: 58170, updated on 12-Aug-2019

Gene summary

Official Symbol Asic5 provided by MGI Official Full Name acid-sensing (proton-gated) ion channel family member 5 provided by MGI Primary source MGI:MGI:1929259 See related Ensembl:ENSMUSG00000028008 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 Inac; Accn5; Blinac Expression Low expression observed in reference dataset See more Orthologs human all

Genomic context

Location: 3; 3 E3 See Asic5 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (81982293..82021233)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (81800844..81825155)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 3 transcripts

Gene: Asic5 ENSMUSG00000028008

Description acid-sensing (proton-gated) ion channel family member 5 [Source:MGI Symbol;Acc:MGI:1929259] Gene Synonyms Accn5, BLINaC, brain-liver-intestine amiloride-sensitive sodium channel Location Chromosome 3: 81,982,290-82,021,233 forward strand. GRCm38:CM000996.2 About this gene This gene has 3 transcripts (splice variants), 131 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Asic5-201 ENSMUST00000029641.9 1657 495aa ENSMUSP00000029641.3 Protein coding CCDS17428 Q9R0Y1 TSL:1 GENCODE basic APPRIS P2

Asic5-202 ENSMUST00000107736.1 1706 453aa ENSMUSP00000103364.1 Protein coding - D3Z0S5 TSL:1 GENCODE basic APPRIS ALT2

Asic5-203 ENSMUST00000142889.1 716 No protein - lncRNA - - TSL:5

58.94 kb Forward strand 81.98Mb 81.99Mb 82.00Mb 82.01Mb 82.02Mb 82.03Mb Genes (Comprehensive set... Asic5-203 >lncRNA

Asic5-201 >protein coding

Asic5-202 >protein coding

Contigs < AC159260.3 AC131757.3 > Genes < Tdo2-201protein coding (Comprehensive set...

< Tdo2-204protein coding

Regulatory Build

81.98Mb 81.99Mb 82.00Mb 82.01Mb 82.02Mb 82.03Mb Reverse strand 58.94 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000029641

24.31 kb Forward strand

Asic5-201 >protein coding

ENSMUSP00000029... Transmembrane heli... Low complexity (Seg) Prints Epithelial sodium channel Pfam Epithelial sodium channel PANTHER Epithelial sodium channel

PTHR11690:SF126 Gene3D 2.60.470.10

1.10.3590.10 1.10.287.820

1.10.287.770

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

Variant Legend stop gained missense variant stop retained variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 495

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

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