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Mouse Gna14 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gna14 conditional 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 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Gna14 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-254N3 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 29.11% 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: 65042 bp, and the size of intron 3 for 3'-loxP site insertion: 4169 bp. The size of effective cKO region: ~655 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 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Gna14 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(7155bp) | A(27.98% 2002) | C(23.1% 1653) | T(25.81% 1847) | G(23.1% 1653)

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% chr19 + 16595691 16598690 3000 browser details YourSeq 153 931 1130 3000 96.5% chr14 - 30465478 30466162 685 browser details YourSeq 140 973 1143 3000 95.7% chr10 + 108316986 108317316 331 browser details YourSeq 104 944 1141 3000 84.1% chr1 + 101400789 101400926 138 browser details YourSeq 78 989 1076 3000 97.8% chr16 - 38961269 38961719 451 browser details YourSeq 77 972 1069 3000 96.7% chr5 + 115806243 115806796 554 browser details YourSeq 65 918 998 3000 91.3% chr3 + 77748166 77748716 551 browser details YourSeq 64 967 1072 3000 92.3% chr10 - 115182361 115182473 113 browser details YourSeq 52 980 1078 3000 77.8% chr2 - 61788623 61788679 57 browser details YourSeq 49 968 1112 3000 98.2% chr10 - 89386113 89386289 177 browser details YourSeq 41 1008 1099 3000 68.2% chr3 - 116483658 116483701 44 browser details YourSeq 38 1401 1876 3000 62.8% chr9 - 21419142 21419535 394 browser details YourSeq 38 1077 1120 3000 97.5% chr16 - 38961271 38961421 151 browser details YourSeq 36 2751 2806 3000 77.5% chr18 - 11043027 11043072 46 browser details YourSeq 33 1109 1141 3000 100.0% chr7 - 83056054 83056086 33 browser details YourSeq 31 1099 1132 3000 97.1% chr10 - 115182361 115182416 56 browser details YourSeq 31 990 1026 3000 97.1% chr7 + 80949302 80949364 63 browser details YourSeq 28 985 1012 3000 100.0% chr7 - 83055987 83056014 28 browser details YourSeq 28 1592 1630 3000 93.6% chr5 + 81554507 81554545 39 browser details YourSeq 25 2760 2786 3000 96.3% chr11 - 89087160 89087186 27

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% chr19 + 16599346 16602345 3000 browser details YourSeq 449 2276 2938 3000 88.8% chr5 - 77285786 77286432 647 browser details YourSeq 437 2282 2941 3000 90.5% chr9 - 51019627 51020287 661 browser details YourSeq 422 2251 2941 3000 87.1% chr2 - 95547851 95548555 705 browser details YourSeq 405 2270 2941 3000 90.0% chr15 - 36439299 36440024 726 browser details YourSeq 390 2270 2893 3000 88.8% chr2 - 13586790 13587413 624 browser details YourSeq 359 2284 2883 3000 89.5% chr2 - 132392196 132392965 770 browser details YourSeq 54 2937 3000 3000 93.6% chr10 + 124752431 124752503 73 browser details YourSeq 52 2940 2998 3000 89.5% chr2 - 163137616 163137672 57 browser details YourSeq 52 2930 3000 3000 83.7% chr15 - 58136028 58136093 66 browser details YourSeq 52 2940 3000 3000 86.3% chr11 - 29566598 29566655 58 browser details YourSeq 51 2942 3000 3000 91.3% chr14 - 51014069 51014126 58 browser details YourSeq 51 2945 3000 3000 90.8% chr1 - 136633413 136633466 54 browser details YourSeq 51 2943 3000 3000 89.3% chr13 + 73931068 73931123 56 browser details YourSeq 50 2944 3000 3000 89.1% chr13 - 56028068 56028122 55 browser details YourSeq 50 2944 3000 3000 89.1% chr11 - 115742214 115742268 55 browser details YourSeq 50 2941 3000 3000 93.2% chr19 + 5788399 5788458 60 browser details YourSeq 50 2944 3000 3000 89.1% chr1 + 177617154 177617208 55 browser details YourSeq 49 2943 3000 3000 87.5% chr15 - 94778323 94778378 56 browser details YourSeq 49 2945 3000 3000 88.9% chr15 - 34271899 34271952 54

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: 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

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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

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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.

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