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Mouse Gna14 Knockout Project (CRISPR/Cas9)

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Mouse Gna14 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Gna14 Knockout Project (CRISPR/Cas9) Objective: To create a Gna14 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Gna14 gene (NCBI Reference Sequence: NM_008137 ; Ensembl: ENSMUSG00000024697 ) is located on Mouse chromosome 19. 7 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000025602). Exon 3~6 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: Exon 3 starts from about 29.11% of the coding region. Exon 3~6 covers 53.33% of the coding region. The size of effective KO region: ~9197 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 6 7 Legends Exon of mouse Gna14 Knockout region Page 2 of 8 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 3 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 1203 bp section downstream of Exon 6 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.6% 492) | C(24.85% 497) | T(25.75% 515) | G(24.8% 496) Note: The 2000 bp section upstream 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(1203bp) | A(27.27% 328) | C(24.69% 297) | T(26.35% 317) | G(21.7% 261) Note: The 1203 bp section downstream of Exon 6 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 8 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% chr19 + 16596941 16598940 2000 browser details YourSeq 38 151 626 2000 62.8% chr9 - 21419142 21419535 394 browser details YourSeq 36 1501 1556 2000 77.5% chr18 - 11043027 11043072 46 browser details YourSeq 32 986 1037 2000 94.3% chr1 + 127354371 127354845 475 browser details YourSeq 28 583 613 2000 96.8% chr1 - 25613441 25613473 33 browser details YourSeq 28 342 380 2000 93.6% chr5 + 81554507 81554545 39 browser details YourSeq 28 577 619 2000 83.4% chr1 + 69656080 69656119 40 browser details YourSeq 27 1527 1555 2000 96.6% chr1 - 36513806 36513834 29 browser details YourSeq 21 571 593 2000 95.7% chr1 + 35954349 35954371 23 Note: The 2000 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 1203 1 1203 1203 100.0% chr19 + 16608138 16609340 1203 browser details YourSeq 49 814 889 1203 84.4% chrX + 166437652 166437726 75 browser details YourSeq 48 824 1028 1203 77.2% chr8 + 26573568 26573760 193 browser details YourSeq 33 846 889 1203 94.6% chr8 - 127968751 127968796 46 browser details YourSeq 29 848 889 1203 85.3% chr9 - 85650186 85650226 41 browser details YourSeq 29 872 923 1203 96.9% chr2 - 170582438 170582489 52 browser details YourSeq 29 845 889 1203 94.0% chr1 + 156685065 156685111 47 browser details YourSeq 29 913 949 1203 89.2% chr1 + 91718840 91718876 37 browser details YourSeq 28 918 950 1203 96.7% chr2 - 28053765 28053806 42 browser details YourSeq 27 910 946 1203 86.5% chr1 - 128673476 128673512 37 browser details YourSeq 27 781 813 1203 83.4% chr1 - 78518160 78518190 31 browser details YourSeq 27 811 864 1203 89.7% chr1 + 77958573 77958625 53 browser details YourSeq 26 846 877 1203 77.8% chr1 - 73047265 73047291 27 browser details YourSeq 25 926 950 1203 100.0% chr1 + 34719211 34719235 25 browser details YourSeq 24 845 868 1203 100.0% chr10 - 30751495 30751518 24 browser details YourSeq 22 929 950 1203 100.0% chr6 - 45742540 45742561 22 browser details YourSeq 22 1139 1160 1203 100.0% chr5 + 131520441 131520462 22 browser details YourSeq 21 503 523 1203 100.0% chr4 - 37586851 37586871 21 browser details YourSeq 21 214 234 1203 100.0% chr11 - 52404946 52404966 21 browser details YourSeq 21 1121 1141 1203 100.0% chr11 - 18948516 18948536 21 Note: The 1203 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Gna14 guanine nucleotide binding protein, alpha 14 [ Mus musculus (house mouse) ] Gene ID: 14675, updated on 13-Aug-2019 Gene summary Official Symbol Gna14 provided by MGI Official Full Name guanine nucleotide binding protein, alpha 14 provided by MGI Primary source MGI:MGI:95769 See related Ensembl:ENSMUSG00000024697 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 AU023208 Expression Biased expression in colon adult (RPKM 20.8), adrenal adult (RPKM 12.1) and 9 other tissues See more Genomic context Location: 19 A; 19 11.29 cM See Gna14 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (16435667..16610818) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (16510157..16685308) Chromosome 19 - NC_000085.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Gna14 ENSMUSG00000024697 Description guanine nucleotide binding protein, alpha 14 [Source:MGI Symbol;Acc:MGI:95769] Gene Synonyms G alpha 14 Location Chromosome 19: 16,435,762-16,610,820 forward strand. GRCm38:CM001012.2 About this gene This gene has 2 transcripts (splice variants), 239 orthologues, 15 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Gna14-201 ENSMUST00000025602.3 3290 355aa ENSMUSP00000025602.2 Protein coding CCDS29685 P30677 TSL:1 GENCODE basic APPRIS P1 Gna14-202 ENSMUST00000237350.1 1377 301aa ENSMUSP00000158560.1 Protein coding - A0A494BBL5 GENCODE basic 195.06 kb Forward strand 16.45Mb 16.50Mb 16.55Mb 16.60Mb Genes (Comprehensive set... Gna14-201 >protein coding Gna14-202 >protein coding Contigs < AC126031.2 AC135377.3 > Genes < Gm8222-201processed pseudogene < Gm50363-201processed pseudogene (Comprehensive set... < Vps13a-205nonsense mediated decay < Vps13a-201protein coding < Vps13a-202lncRNA Regulatory Build 16.45Mb 16.50Mb 16.55Mb 16.60Mb Reverse strand 195.06 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 RNA gene processed transcript pseudogene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000025602 175.06 kb Forward strand Gna14-201 >protein coding ENSMUSP00000025... Coiled-coils (Ncoils) Superfamily G protein alpha subunit, helical insertion P-loop containing nucleoside triphosphate hydrolase SMART Guanine nucleotide binding protein (G-protein), alpha subunit Prints G-protein alpha subunit, group Q Guanine nucleotide binding protein (G-protein), alpha subunit Pfam Guanine nucleotide binding protein (G-protein), alpha subunit PANTHER PTHR10218:SF213 Guanine nucleotide binding protein (G-protein), alpha subunit Gene3D 3.40.50.300 G protein alpha subunit, helical insertion CDD Guanine nucleotide binding protein (G-protein), alpha subunit All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 355 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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