https://www.alphaknockout.com
Mouse Cacna1c Knockout Project (CRISPR/Cas9)
Objective: To create a Cacna1c knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Cacna1c gene (NCBI Reference Sequence: NM_001256002 ; Ensembl: ENSMUSG00000051331 ) is located on Mouse chromosome 6. 48 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 48 (Transcript: ENSMUST00000112793). Exon 7 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for mutations that inactivate the gene do not survive to term. Selective ablation in beta cells resulted in impaired insulin secretion and systemic glucose intolerance. Heterozygotes were hypoactive, showed increased anxiety, and poor motor coordination.
Exon 7 starts from about 13.76% of the coding region. Exon 7 covers 2.96% of the coding region. The size of effective KO region: ~197 bp. The KO 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 7 48
Legends Exon of mouse Cacna1c Knockout region
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Overview of the Dot Plot (up) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 bp section upstream of Exon 7 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 Dot Plot (down) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 bp section downstream of Exon 7 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.
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Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(25.9% 518) | C(22.85% 457) | T(30.2% 604) | G(21.05% 421)
Note: The 2000 bp section upstream of Exon 7 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.
Overview of the GC Content Distribution (down) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(24.85% 497) | C(24.65% 493) | T(27.95% 559) | G(22.55% 451)
Note: The 2000 bp section downstream of Exon 7 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 2000 1 2000 2000 100.0% chr6 - 118751566 118753565 2000 browser details YourSeq 29 1957 1991 2000 93.8% chr7 - 82520220 82520256 37 browser details YourSeq 29 294 347 2000 87.9% chr1 - 118346050 118346102 53 browser details YourSeq 23 400 422 2000 100.0% chr2 - 60307984 60308006 23 browser details YourSeq 22 1460 1481 2000 100.0% chr5 - 145649936 145649957 22 browser details YourSeq 21 1545 1565 2000 100.0% chr13 - 20663351 20663371 21 browser details YourSeq 21 1428 1448 2000 100.0% chr7 + 89785951 89785971 21 browser details YourSeq 21 1911 1931 2000 100.0% chr12 + 89678896 89678916 21 browser details YourSeq 20 1002 1021 2000 100.0% chr1 + 38527462 38527481 20
Note: The 2000 bp section upstream of Exon 7 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 2000 1 2000 2000 100.0% chr6 - 118749369 118751368 2000 browser details YourSeq 42 629 1161 2000 63.9% chr2 + 76443595 76443996 402 browser details YourSeq 21 661 681 2000 100.0% chr5 - 77491556 77491576 21 browser details YourSeq 20 1101 1120 2000 100.0% chr1 + 9853075 9853094 20
Note: The 2000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Cacna1c calcium channel, voltage-dependent, L type, alpha 1C subunit [ Mus musculus (house mouse) ] Gene ID: 12288, updated on 15-Oct-2019
Gene summary
Official Symbol Cacna1c provided by MGI Official Full Name calcium channel, voltage-dependent, L type, alpha 1C subunit provided by MGI Primary source MGI:MGI:103013 See related Ensembl:ENSMUSG00000051331 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 MBC; Cav1.2; Cchl1a1; MELC-CC; D930026N18Rik Expression Broad expression in heart adult (RPKM 11.2), adrenal adult (RPKM 6.3) and 16 other tissues See more Orthologs human all
Genomic context
Location: 6 F1; 6 55.86 cM See Cacna1c in Genome Data Viewer Exon count: 53
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (118587240..119197384, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (118542314..119146427, complement)
Chromosome 6 - NC_000072.6
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Transcript information: This gene has 27 transcripts
Gene: Cacna1c ENSMUSG00000051331
Description calcium channel, voltage-dependent, L type, alpha 1C subunit [Source:MGI Symbol;Acc:MGI:103013] Gene Synonyms (alpha)1 subunit, Cav1.2, Cchl1a1, D930026N18Rik, L-type Cav1.2 Location Chromosome 6: 118,587,240-119,196,418 reverse strand. GRCm38:CM000999.2 About this gene This gene has 27 transcripts (splice variants), 216 orthologues, 26 paralogues, is a member of 1 Ensembl protein family and is associated with 30 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Cacna1c- ENSMUST00000186889.6 13162 2169aa ENSMUSP00000140056.1 Protein coding CCDS80592 Q0PCR6 TSL:1 207 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000078320.13 8062 2139aa ENSMUSP00000077433.8 Protein coding CCDS80590 Q01815 TSL:1 202 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000112790.8 7631 2139aa ENSMUSP00000108410.3 Protein coding CCDS80589 F8WJL1 TSL:1 203 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000075591.12 7584 2139aa ENSMUSP00000075021.7 Protein coding CCDS80590 Q01815 TSL:1 201 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000112825.8 7577 1869aa ENSMUSP00000108444.2 Protein coding CCDS71821 Q01815 TSL:1 205 GENCODE basic
Cacna1c- ENSMUST00000187940.6 7507 2169aa ENSMUSP00000141033.1 Protein coding CCDS80593 C7TQ57 TSL:1 212 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000187474.6 6911 2169aa ENSMUSP00000140961.1 Protein coding CCDS80594 C7TQ59 TSL:1 210 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000112793.9 6669 2222aa ENSMUSP00000108413.4 Protein coding CCDS80587 F7C376 TSL:1 204 GENCODE basic
Cacna1c- ENSMUST00000190285.1 6585 2194aa ENSMUSP00000141015.1 Protein coding CCDS80595 A0A087WSE7 TSL:1 222 GENCODE basic APPRIS P4
Cacna1c- ENSMUST00000188106.6 6462 2153aa ENSMUSP00000140886.1 Protein coding CCDS80588 A0A087WS40 TSL:1 214 GENCODE basic
Cacna1c- ENSMUST00000220022.1 7765 2063aa ENSMUSP00000151458.1 Protein coding - A0A1W2P731 TSL:5 227 GENCODE basic
Cacna1c- ENSMUST00000219833.1 7591 2005aa ENSMUSP00000151462.1 Protein coding - A0A1W2P6Z7 TSL:5 226 GENCODE basic
Cacna1c- ENSMUST00000219018.1 7516 1980aa ENSMUSP00000151863.1 Protein coding - A0A1W2P805 TSL:5 224 GENCODE basic Page 7 of 10 https://www.alphaknockout.com
Cacna1c- ENSMUST00000219223.1 7483 1969aa ENSMUSP00000151391.1 Protein coding - A0A1W2P6T3 TSL:5 225 GENCODE basic
Cacna1c- ENSMUST00000188522.6 7338 2164aa ENSMUSP00000140920.1 Protein coding - A0A087WS67 TSL:5 216 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000187386.6 6906 2135aa ENSMUSP00000140341.1 Protein coding - A0A087WQT9 TSL:5 209 GENCODE basic
Cacna1c- ENSMUST00000187317.6 6663 2187aa ENSMUSP00000140693.1 Protein coding - A0A087WRM3 TSL:5 208 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000189389.6 6603 2167aa ENSMUSP00000139855.1 Protein coding - A0A087WPP1 TSL:5 219 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000185345.6 6579 2159aa ENSMUSP00000140833.1 Protein coding - A0A087WRZ6 TSL:5 206 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000189520.6 6570 2156aa ENSMUSP00000140220.1 Protein coding - A0A087WQJ4 TSL:5 220 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000188078.6 6519 2139aa ENSMUSP00000140415.1 Protein coding - A0A087WR02 TSL:5 213 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000188865.6 6519 2139aa ENSMUSP00000139981.1 Protein coding - A0A087WPZ8 TSL:5 217 GENCODE basic APPRIS ALT2
Cacna1c- ENSMUST00000189930.1 4931 No - Retained - - TSL:NA 221 protein intron
Cacna1c- ENSMUST00000188181.6 7577 No - lncRNA - - TSL:1 215 protein
Cacna1c- ENSMUST00000191196.1 2374 No - lncRNA - - TSL:1 223 protein
Cacna1c- ENSMUST00000189057.1 954 No - lncRNA - - TSL:1 218 protein
Cacna1c- ENSMUST00000187849.1 715 No - lncRNA - - TSL:3 211 protein
629.18 kb Forward strand 118.6Mb 118.8Mb 119.0Mb 119.2Mb Genes Gm25905-201 >snRNA Dcp1b-205 >retained intron (Comprehensive set...
Dcp1b-204 >retained intron
Dcp1b-202 >protein coding
Dcp1b-201 >protein coding
Contigs < AC036121.11 < AC127328.6 < AC163353.3 < AC126453.5 Genes (Comprehensive set... < Cacna1c-207protein coding
< Cacna1c-223lncRNA < Cacna1c-221retained intron < Cacna1c-218lncRNA
< Cacna1c-224protein coding
< Cacna1c-225protein coding Page 8 of 10
< Cacna1c-227protein coding
< Cacna1c-226protein coding
< Cacna1c-205protein coding
< Cacna1c-215lncRNA
< Cacna1c-202protein coding
< Cacna1c-203protein coding
< Cacna1c-201protein coding
< Cacna1c-212protein coding
< Cacna1c-210protein coding
< Cacna1c-216protein coding
< Cacna1c-217protein coding
< Cacna1c-206protein coding
< Cacna1c-208protein coding
< Cacna1c-220protein coding
< Cacna1c-219protein coding
< Cacna1c-213protein coding
< Cacna1c-209protein coding
< Cacna1c-222protein coding
< Cacna1c-214protein coding
< Cacna1c-204protein coding
< Cacna1c-211lncRNA
< Gm18981-201processed pseudogene
Regulatory Build
118.6Mb 118.8Mb 119.0Mb 119.2Mb Reverse strand 629.18 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
processed transcript RNA gene pseudogene 629.18 kb Forward strand 118.6Mb 118.8Mb 119.0Mb 119.2Mb Genes Gm25905-201 >snRNA Dcp1b-205 >retained intron (Comprehensive set...
Dcp1b-204 >retained intron
Dcp1b-202 >protein coding
Dcp1b-201 >protein coding
Contigs < AC036121.11 < AC127328.6 < AC163353.3 < AC126453.5 Genes (Comprehensive set... < Cacna1c-207protein coding
< Cacna1c-223lncRNA < Cacna1c-221retained intron < Cacna1c-218lncRNA
< Cacna1c-224protein coding https://www.alphaknockout.com
< Cacna1c-225protein coding
< Cacna1c-227protein coding
< Cacna1c-226protein coding
< Cacna1c-205protein coding
< Cacna1c-215lncRNA
< Cacna1c-202protein coding
< Cacna1c-203protein coding
< Cacna1c-201protein coding
< Cacna1c-212protein coding
< Cacna1c-210protein coding
< Cacna1c-216protein coding
< Cacna1c-217protein coding
< Cacna1c-206protein coding
< Cacna1c-208protein coding
< Cacna1c-220protein coding
< Cacna1c-219protein coding
< Cacna1c-213protein coding
< Cacna1c-209protein coding
< Cacna1c-222protein coding
< Cacna1c-214protein coding
< Cacna1c-204protein coding
< Cacna1c-211lncRNA
< Gm18981-201processed pseudogene
Regulatory Build
118.6Mb 118.8Mb 119.0Mb 119.2Mb Reverse strand 629.18 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
processed transcript RNA gene pseudogene
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Transcript: ENSMUST00000112793
< Cacna1c-204protein coding
Reverse strand 514.08 kb
ENSMUSP00000108... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF81324 SMART Voltage-dependent calcium channel, alpha-1 subunit, IQ domain Prints Voltage-dependent calcium channel, L-type, alpha-1C subunit
Voltage-dependent calcium channel, alpha-1 subunit
Voltage-dependent calcium channel, L-type, alpha-1 subunit Pfam Ion transport domain Voltage-gated calcium channel subunit alpha, C-terminal
Voltage-dependent calcium channel, alpha-1 subunit, IQ domain
Voltage-dependent L-type calcium channel, IQ-associated domain PANTHER PTHR45628
PTHR45628:SF11 Gene3D Voltage-dependent channel domain superfamily 1.10.238.10
1.10.287.70
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2222
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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