https://www.alphaknockout.com
Mouse Chrna1 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Chrna1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Chrna1 gene (NCBI Reference Sequence: NM_007389 ; Ensembl: ENSMUSG00000027107 ) is located on Mouse chromosome 2. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000028515). 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 Chrna1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-93D24 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 neonatal lethality, kyphosis, carpotosis, absent miniature and nerve- evoked endplant potential, increased motor neuron number, and abnormal neuromuscular synapse.
Exon 5 starts from about 25.16% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1651 bp, and the size of intron 5 for 3'-loxP site insertion: 768 bp. The size of effective cKO region: ~696 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 4 5 6 9 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Chrna1 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(7196bp) | A(25.65% 1846) | C(23.96% 1724) | T(27.78% 1999) | G(22.61% 1627)
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% chr2 - 73571859 73574858 3000 browser details YourSeq 120 705 941 3000 93.7% chr10 - 118433577 118433876 300 browser details YourSeq 115 717 942 3000 93.1% chr15 + 49753638 49754285 648 browser details YourSeq 102 723 930 3000 81.9% chr1 - 125514424 125514563 140 browser details YourSeq 102 700 933 3000 85.8% chr12 + 27229621 27229838 218 browser details YourSeq 100 706 908 3000 93.4% chr14 - 65312045 65489096 177052 browser details YourSeq 97 725 922 3000 92.4% chr10 + 116471549 116846601 375053 browser details YourSeq 93 801 939 3000 95.3% chr10 + 71035870 71183017 147148 browser details YourSeq 92 672 930 3000 94.3% chr1 + 192210169 192210460 292 browser details YourSeq 89 711 921 3000 80.2% chr1 - 125514364 125514490 127 browser details YourSeq 87 720 939 3000 82.9% chr8 - 13619752 13619928 177 browser details YourSeq 87 715 926 3000 82.7% chr1 - 190182855 190183042 188 browser details YourSeq 85 732 914 3000 94.9% chr10 - 112223214 112223717 504 browser details YourSeq 85 717 900 3000 94.8% chr10 + 19198668 19198926 259 browser details YourSeq 84 715 939 3000 93.7% chr13 + 77455922 77456177 256 browser details YourSeq 82 683 886 3000 80.7% chr11 + 77740205 77740331 127 browser details YourSeq 79 710 935 3000 79.2% chr8 + 113786065 113786191 127 browser details YourSeq 74 719 922 3000 77.2% chr4 - 17743770 17743932 163 browser details YourSeq 74 719 921 3000 81.6% chr11 - 100194146 100194335 190 browser details YourSeq 74 696 882 3000 83.0% chr10 - 41645562 41645725 164
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% chr2 - 73568163 73571162 3000 browser details YourSeq 128 20 172 3000 93.3% chr17 + 26931104 26931438 335 browser details YourSeq 128 1 174 3000 85.6% chr13 + 100545254 100545418 165 browser details YourSeq 125 22 192 3000 84.4% chr5 + 107390576 107390728 153 browser details YourSeq 119 20 173 3000 93.5% chr11 - 97846585 97846907 323 browser details YourSeq 119 22 171 3000 87.0% chr1 + 100110565 100110710 146 browser details YourSeq 118 20 172 3000 88.1% chr1 - 135418836 135418984 149 browser details YourSeq 116 8 170 3000 91.5% chr8 + 120615296 120615524 229 browser details YourSeq 116 20 172 3000 85.3% chr15 + 12296899 12297047 149 browser details YourSeq 115 20 174 3000 85.5% chr18 - 53206168 53206319 152 browser details YourSeq 115 20 167 3000 86.2% chr16 - 36101035 36101178 144 browser details YourSeq 115 20 171 3000 85.2% chr8 + 125606557 125606704 148 browser details YourSeq 115 20 171 3000 89.2% chr6 + 108681923 108682072 150 browser details YourSeq 114 36 172 3000 88.8% chrX - 103257396 103257528 133 browser details YourSeq 113 20 167 3000 85.5% chr13 - 38046632 38046775 144 browser details YourSeq 113 37 192 3000 85.3% chr1 + 44572660 44572809 150 browser details YourSeq 112 37 171 3000 88.6% chr4 - 135295546 135295676 131 browser details YourSeq 112 20 172 3000 83.9% chr19 + 30531995 30532143 149 browser details YourSeq 110 36 170 3000 87.8% chr9 - 83757608 83757738 131 browser details YourSeq 110 20 168 3000 88.3% chr6 - 88438461 88438785 325
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: Chrna1 cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) [ Mus musculus (house mouse) ] Gene ID: 11435, updated on 11-Sep-2019
Gene summary
Official Symbol Chrna1 provided by MGI Official Full Name cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) provided by MGI Primary source MGI:MGI:87885 See related Ensembl:ENSMUSG00000027107 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Acra; Achr-1; AI385656; AI608266 Summary This gene encodes an alpha subunit of the muscle-derived nicotinic acetylcholine receptor, a pentameric neurotransmitter Expression receptor and member of the ligand-gated ion channel superfamily. The alpha subunit plays a role in substrate binding and channel gating. [provided by RefSeq, Nov 2012] Orthologs Biased expression in limb E14.5 (RPKM 3.2), testis adult (RPKM 1.0) and 4 other tissues See more human all
Genomic context
Location: 2 C3; 2 43.76 cM See Chrna1 in Genome Data Viewer
Exon count: 9
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (73563281..73580338, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (73401338..73418363, complement)
Chromosome 2 - NC_000068.7
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Transcript information: This gene has 1 transcript
Gene: Chrna1 ENSMUSG00000027107
Description cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) [Source:MGI Symbol;Acc:MGI:87885] Gene Synonyms Achr-1, Acra Location Chromosome 2: 73,563,215-73,580,338 reverse strand. GRCm38:CM000995.2 About this gene This gene has 1 transcript (splice variant), 242 orthologues, 41 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
Chrna1-201 ENSMUST00000028515.3 4386 457aa ENSMUSP00000028515.3 Protein coding CCDS16132 P04756 Q05A24 TSL:1 GENCODE basic APPRIS P1
37.12 kb Forward strand
73.56Mb 73.57Mb 73.58Mb 73.59Mb Genes Chrna1os-201 >lncRNA (Comprehensive set...
Chrna1os-202 >lncRNA
Contigs AL928813.10 > AL928889.14 >
Genes < Chrna1-201protein coding (Comprehensive set...
Regulatory Build
73.56Mb 73.57Mb 73.58Mb 73.59Mb Reverse strand 37.12 kb
Regulation Legend
CTCF Enhancer Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
merged Ensembl/Havana
Non-Protein Coding
RNA gene
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Transcript: ENSMUST00000028515
< Chrna1-201protein coding
Reverse strand 17.12 kb
ENSMUSP00000028... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Cleavage site (Sign... TIGRFAM Neurotransmitter-gated ion-channel Superfamily Neurotransmitter-gated ion-channel transmembrane domain superfamily
Neurotransmitter-gated ion-channel ligand-binding domain superfamily Prints Nicotinic acetylcholine receptor
Neurotransmitter-gated ion-channel Pfam Neurotransmitter-gated ion-channel ligand-binding domain Neurotransmitter-gated ion-channel transmembrane domain
PROSITE patterns Neurotransmitter-gated ion-channel, conserved site PANTHER PTHR18945:SF74
Neurotransmitter-gated ion-channel Gene3D Neurotransmitter-gated ion-channel ligand-binding domain superfamily
1.20.58.390 CDD cd19014 cd19064
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 40 80 120 160 200 240 280 320 360 400 457
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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