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Mouse Rcan1 Knockout Project (CRISPR/Cas9)

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Mouse Rcan1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rcan1 Knockout Project (CRISPR/Cas9) Objective: To create a Rcan1 knockout Mouse model (C57BL/6J ) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rcan1 gene (NCBI Reference Sequence: NM_001081549 ; Ensembl: ENSMUSG00000022951 ) is located on Mouse chromosome 16. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000060005). Exon 2~3 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: Unstressed homozygous mutant mice show no overt phenotype other than a slight reduction in heart size and an impaired T helper 1 response. Stress-induced cardiac hypertrophy, however, is attenuated in mutant mice. Exon 2 starts from about 32.8% of the coding region. Exon 2~3 covers 44.36% of the coding region. The size of effective KO region: ~2390 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 Legends Exon of mouse Rcan1 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 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 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.15% 503) | C(25.2% 504) | T(26.65% 533) | G(23.0% 460) Note: The 2000 bp section upstream of Exon 2 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(23.95% 479) | C(27.35% 547) | T(22.65% 453) | G(26.05% 521) Note: The 2000 bp section downstream of Exon 3 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. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr16 - 92397437 92399436 2000 browser details YourSeq 28 869 898 2000 89.7% chr2 + 156483908 156483936 29 browser details YourSeq 24 778 801 2000 100.0% chr13 + 119410558 119410581 24 browser details YourSeq 23 470 492 2000 100.0% chr11 - 88987540 88987562 23 browser details YourSeq 22 1502 1523 2000 100.0% chr14 - 25824891 25824912 22 browser details YourSeq 21 1166 1186 2000 100.0% chr10 + 94675141 94675161 21 browser details YourSeq 20 872 891 2000 100.0% chr13 + 97007734 97007753 20 Note: The 2000 bp section upstream of Exon 2 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% chr16 - 92393866 92395865 2000 browser details YourSeq 65 1614 1679 2000 100.0% chr6 + 124639146 124639212 67 browser details YourSeq 58 1620 1677 2000 100.0% chr1 - 123052310 123052367 58 browser details YourSeq 40 1640 1679 2000 100.0% chr1 - 124125895 124125934 40 browser details YourSeq 38 1731 1786 2000 95.3% chr12 + 83546066 83546276 211 browser details YourSeq 27 1721 1751 2000 82.2% chr16 + 93881755 93881782 28 browser details YourSeq 26 1740 1769 2000 89.3% chr11 + 115341341 115341369 29 browser details YourSeq 25 1727 1751 2000 100.0% chr10 - 61706796 61706820 25 browser details YourSeq 24 1737 1769 2000 88.5% chr11 - 62675561 62675592 32 browser details YourSeq 23 1468 1490 2000 100.0% chr10 - 81384649 81384671 23 browser details YourSeq 23 468 490 2000 100.0% chr18 + 87938491 87938513 23 browser details YourSeq 23 1842 1865 2000 100.0% chr1 + 8704381 8704406 26 browser details YourSeq 21 1727 1747 2000 100.0% chr6 - 113806686 113806706 21 browser details YourSeq 20 448 467 2000 100.0% chr1 - 72313735 72313754 20 browser details YourSeq 20 468 487 2000 100.0% chr1 - 28800782 28800801 20 browser details YourSeq 20 468 487 2000 100.0% chr1 - 5487319 5487338 20 browser details YourSeq 20 468 487 2000 100.0% chr1 + 167139745 167139764 20 browser details YourSeq 20 1496 1515 2000 100.0% chr1 + 139228241 139228260 20 browser details YourSeq 20 468 487 2000 100.0% chr1 + 30627863 30627882 20 browser details YourSeq 20 468 487 2000 100.0% chr1 + 23697735 23697754 20 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Rcan1 regulator of calcineurin 1 [ Mus musculus (house mouse) ] Gene ID: 54720, updated on 21-Aug-2019 Gene summary Official Symbol Rcan1 provided by MGI Official Full Name regulator of calcineurin 1 provided by MGI Primary source MGI:MGI:1890564 See related Ensembl:ENSMUSG00000022951 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 CSP1; DSC1; RCN1; Dscr1; MCIP1; CALP1L; Adapt78 Expression Ubiquitous expression in CNS E18 (RPKM 17.8), heart adult (RPKM 14.9) and 28 other tissues See more Orthologs human all Genomic context Location: 16 C4; 16 53.6 cM See Rcan1 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (92391951..92466169, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (92392198..92466391, complement) Chromosome 16 - NC_000082.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Rcan1 ENSMUSG00000022951 Description regulator of calcineurin 1 [Source:MGI Symbol;Acc:MGI:1890564] Gene Synonyms 2410048A02Rik, ADAPT78, CSP1, Dscr1, MCIP1 Location Chromosome 16: 92,391,953-92,470,867 reverse strand. GRCm38:CM001009.2 About this gene This gene has 7 transcripts (splice variants), 201 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 21 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Rcan1- ENSMUST00000060005.14 2324 251aa ENSMUSP00000060394.8 Protein coding CCDS37405 Q9JHG6 TSL:1 202 GENCODE basic Rcan1- ENSMUST00000023672.9 2190 198aa ENSMUSP00000023672.7 Protein coding CCDS28337 Q542V6 TSL:1 201 Q9JHG6 GENCODE basic APPRIS P1 Rcan1- ENSMUST00000232239.1 2319 198aa ENSMUSP00000156053.1 Protein coding - Q9JHG6 GENCODE 206 basic Rcan1- ENSMUST00000231410.1 2222 118aa ENSMUSP00000156110.1 Protein coding - Q9JHG6 GENCODE 203 basic Rcan1- ENSMUST00000232197.1 1506 118aa ENSMUSP00000155863.1 Protein coding - Q9JHG6 GENCODE 205 basic Rcan1- ENSMUST00000231813.1 440 59aa ENSMUSP00000156351.1 Nonsense mediated - A0A338P7E2 CDS 5' 204 decay incomplete Rcan1- ENSMUST00000232457.1 387 No - lncRNA - - - 207 protein Page 7 of 9 https://www.alphaknockout.com 98.92 kb Forward strand 92.40Mb 92.42Mb 92.44Mb 92.46Mb 92.48Mb Genes Gm46555-201 >lncRNA (Comprehensive set... 2410124H12Rik-201 >lncRNA Contigs < AC162305.5 < AC174448.2 Genes (Comprehensive set... < Gm29805-201lncRNA < Rcan1-207lncRNA < Rcan1-202protein coding < Rcan1-206protein coding < Rcan1-201protein coding < Rcan1-203protein coding < Rcan1-204nonsense mediated decay < Rcan1-205protein coding Regulatory Build 92.40Mb 92.42Mb 92.44Mb 92.46Mb 92.48Mb Reverse strand 98.92 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000060005 < Rcan1-202protein coding Reverse strand 74.19 kb ENSMUSP00000060... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily RNA-binding domain superfamily Pfam Calcipressin PANTHER Calcipressin Calcipressin-1 Gene3D Nucleotide-binding alpha-beta plait domain superfamily CDD Calcipressin-1, RNA recognition motif All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 251 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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