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Mouse Gng12 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gng12 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gng12 gene (NCBI Reference Sequence: NM_025278 ; Ensembl: ENSMUSG00000036402 ) is located on Mouse chromosome 6. 5 exons are identified, with the ATG start codon in exon 4 and the TAG stop codon in exon 5 (Transcript: ENSMUST00000043148). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Gng12 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-107C18 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 4~5 covers 100.0% of the coding region. Start codon is in exon 4, and stop codon is in exon 5. The size of intron 3 for 5'-loxP site insertion: 71847 bp. The size of effective cKO region: ~2109 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele T A

5' gRNA region G 3'

1 4 5

Targeting vector T A G

Targeted allele T A G

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Gng12 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(8336bp) | A(27.16% 2264) | C(22.83% 1903) | T(27.39% 2283) | G(22.62% 1886)

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% chr6 + 67012502 67015501 3000 browser details YourSeq 388 6 1005 3000 95.8% chr11 - 78475416 78585533 110118 browser details YourSeq 364 1 401 3000 95.5% chr7 + 40190952 40191360 409 browser details YourSeq 347 1 395 3000 94.2% chr3 + 137905026 137905431 406 browser details YourSeq 345 14 388 3000 96.3% chrX + 102121664 102127669 6006 browser details YourSeq 337 1 396 3000 93.8% chrX - 98962058 98962461 404 browser details YourSeq 308 1 396 3000 89.9% chr17 - 65560355 65560759 405 browser details YourSeq 303 4 397 3000 90.3% chr14 + 109077976 109078367 392 browser details YourSeq 292 1 395 3000 88.5% chr6 - 3863022 3863454 433 browser details YourSeq 282 41 398 3000 90.6% chr18 + 31738714 31739073 360 browser details YourSeq 259 1 409 3000 85.7% chr1 + 183371093 183371468 376 browser details YourSeq 181 4 395 3000 83.8% chr1 - 60267377 60267616 240 browser details YourSeq 150 33 309 3000 78.8% chr12 - 13201412 13201666 255 browser details YourSeq 34 1633 1686 3000 73.0% chr11 - 72950907 72950947 41 browser details YourSeq 26 913 938 3000 100.0% chr3 - 96380236 96380261 26

Note: The 3000 bp section upstream of Exon 4 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% chr6 + 67017588 67020587 3000 browser details YourSeq 577 1359 1974 3000 97.6% chr12 + 45883022 45883701 680 browser details YourSeq 55 2792 2918 3000 71.7% chr2 + 34161551 34161677 127 browser details YourSeq 46 2796 2919 3000 68.6% chr3 - 146106638 146106761 124 browser details YourSeq 40 2794 2919 3000 97.7% chr1 - 169924907 169925033 127 browser details YourSeq 37 2786 2826 3000 95.2% chr3 - 129980641 129980681 41 browser details YourSeq 34 2784 2819 3000 97.3% chr19 - 9962056 9962091 36 browser details YourSeq 34 2784 2819 3000 97.3% chr17 - 47186234 47186269 36 browser details YourSeq 32 2784 2819 3000 94.5% chr17 - 50185588 50185623 36 browser details YourSeq 31 2784 2818 3000 94.3% chr15 + 73969123 73969157 35 browser details YourSeq 30 2784 2819 3000 91.7% chr1 - 136392201 136392236 36 browser details YourSeq 28 2784 2817 3000 91.2% chr3 - 105535406 105535439 34 browser details YourSeq 26 2794 2821 3000 88.9% chr4 - 11682031 11682057 27 browser details YourSeq 26 2793 2819 3000 100.0% chr14 + 20574609 20574636 28 browser details YourSeq 25 2790 2816 3000 96.3% chr14 + 49021365 49021391 27 browser details YourSeq 22 2791 2816 3000 92.4% chr14 - 75336981 75337006 26 browser details YourSeq 22 1694 1715 3000 100.0% chr1 + 155155579 155155600 22

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: Gng12 guanine nucleotide binding protein (G protein), gamma 12 [ Mus musculus (house mouse) ] Gene ID: 14701, updated on 12-Aug-2019

Gene summary

Official Symbol Gng12 provided by MGI Official Full Name guanine nucleotide binding protein (G protein), gamma 12 provided by MGI Primary source MGI:MGI:1336171 See related Ensembl:ENSMUSG00000036402 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 AA536815; AI115529; AI314170; AI842738; 2010305F15Rik Expression Ubiquitous expression in large intestine adult (RPKM 26.2), kidney adult (RPKM 24.5) and 28 other tissues See more Orthologs human all

Genomic context

Location: 6 C1; 6 30.68 cM See Gng12 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (66896397..67021361)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (66846391..66971355)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 9 transcripts

Gene: Gng12 ENSMUSG00000036402

Description guanine nucleotide binding protein (G protein), gamma 12 [Source:MGI Symbol;Acc:MGI:1336171] Gene Synonyms 2010305F15Rik Location Chromosome 6: 66,896,397-67,021,350 forward strand. GRCm38:CM000999.2 About this gene This gene has 9 transcripts (splice variants), 232 orthologues, 12 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gng12-201 ENSMUST00000043148.12 4248 72aa ENSMUSP00000046557.6 Protein coding CCDS39502 Q9DAS9 TSL:1 GENCODE basic APPRIS P1

Gng12-204 ENSMUST00000114225.7 1876 72aa ENSMUSP00000109863.1 Protein coding CCDS39502 Q9DAS9 TSL:1 GENCODE basic APPRIS P1

Gng12-202 ENSMUST00000114222.3 1354 72aa ENSMUSP00000109860.1 Protein coding CCDS39502 Q9DAS9 TSL:1 GENCODE basic APPRIS P1

Gng12-205 ENSMUST00000114226.7 1155 72aa ENSMUSP00000109864.1 Protein coding CCDS39502 Q9DAS9 TSL:1 GENCODE basic APPRIS P1

Gng12-206 ENSMUST00000114227.7 1125 72aa ENSMUSP00000109865.1 Protein coding CCDS39502 Q9DAS9 TSL:1 GENCODE basic APPRIS P1

Gng12-207 ENSMUST00000114228.7 748 72aa ENSMUSP00000109866.1 Protein coding CCDS39502 Q9DAS9 TSL:1 GENCODE basic APPRIS P1

Gng12-203 ENSMUST00000114224.7 680 72aa ENSMUSP00000109862.1 Protein coding CCDS39502 Q9DAS9 TSL:3 GENCODE basic APPRIS P1

Gng12-208 ENSMUST00000204511.2 739 83aa ENSMUSP00000145346.1 Protein coding - A0A0N4SW28 TSL:5 GENCODE basic

Gng12-209 ENSMUST00000204862.1 380 65aa ENSMUSP00000145234.1 Protein coding - A0A0N4SVT3 CDS 3' incomplete TSL:3

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144.95 kb Forward strand 66.90Mb 66.95Mb 67.00Mb Genes Gng12-201 >protein coding (Comprehensive set...

Gng12-207 >protein coding

Gng12-206 >protein coding

Gng12-205 >protein coding

Gng12-208 >protein coding

Gng12-204 >protein coding

Gng12-203 >protein coding

Gng12-202 >protein coding

Gng12-209 >protein coding

Gm15644-201 >processed pseudogene

Gm29848-201 >processed pseudogene

Gm25260-201 >snRNA

Contigs < AC107650.19 Genes < 4930597O21Rik-202retained intron < Gm36816-202lncRNA (Comprehensive set...

< 4930597O21Rik-201lncRNA < Gm36816-201lncRNA

Regulatory Build

66.90Mb 66.95Mb 67.00Mb Reverse strand 144.95 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000043148

124.95 kb Forward strand

Gng12-201 >protein coding

ENSMUSP00000046... Superfamily G-protein gamma-like domain superfamily

SMART G-protein gamma-like domain

SM01224 Prints G-protein, gamma subunit Pfam G-protein gamma-like domain

PROSITE profiles G-protein gamma-like domain

PANTHER G-protein, gamma subunit

PTHR13809:SF9 Gene3D G-protein gamma-like domain superfamily CDD G-protein gamma-like domain

All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Y Y

Variant Legend

synonymous variant

Scale bar 0 8 16 24 32 40 48 56 64 72

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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