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Mouse Gabrg1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gabrg1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gabrg1 gene (NCBI Reference Sequence: NM_010252 ; Ensembl: ENSMUSG00000001260 ) is located on Mouse chromosome 5. 9 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000031119). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Gabrg1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-241N21 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 2 starts from about 7.53% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 26010 bp, and the size of intron 2 for 3'-loxP site insertion: 21243 bp. The size of effective cKO region: ~649 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 2 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Gabrg1 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(7149bp) | A(31.78% 2272) | C(17.9% 1280) | T(34.19% 2444) | G(16.13% 1153)

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% chr5 - 70816356 70819355 3000 browser details YourSeq 546 785 1557 3000 89.2% chr9 + 109456801 109502957 46157 browser details YourSeq 521 793 1521 3000 91.5% chr1 + 63945115 63945949 835 browser details YourSeq 507 791 1505 3000 91.0% chr6 + 76772436 76773211 776 browser details YourSeq 505 793 1541 3000 90.6% chr8 + 14278850 14279621 772 browser details YourSeq 503 792 1556 3000 88.8% chr10 - 53454116 53454871 756 browser details YourSeq 502 792 1557 3000 89.5% chr7 - 4079584 4080353 770 browser details YourSeq 498 792 1483 3000 91.6% chr13 - 42944737 42945447 711 browser details YourSeq 496 795 1556 3000 89.9% chr12 - 30722216 30722983 768 browser details YourSeq 492 791 1556 3000 86.8% chr7 + 13547682 13548423 742 browser details YourSeq 492 791 1520 3000 89.2% chr17 + 34255764 34256451 688 browser details YourSeq 488 793 1556 3000 90.4% chr3 - 56320662 56321502 841 browser details YourSeq 486 785 1556 3000 90.7% chr2 - 128980833 128981625 793 browser details YourSeq 484 791 1513 3000 90.1% chrX - 165360329 165361047 719 browser details YourSeq 484 792 1552 3000 90.0% chr13 + 78749586 78750390 805 browser details YourSeq 481 797 1556 3000 90.4% chr5 - 13832347 13833122 776 browser details YourSeq 476 792 1488 3000 89.7% chr15 + 69871956 69872772 817 browser details YourSeq 469 793 1513 3000 88.4% chr5 + 109234280 109234959 680 browser details YourSeq 462 799 1493 3000 90.0% chr2 + 117772430 117773186 757 browser details YourSeq 460 793 1521 3000 88.9% chrX - 46753134 47086601 333468

Note: The 3000 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 3000 1 3000 3000 100.0% chr5 - 70812707 70815706 3000 browser details YourSeq 300 43 537 3000 88.1% chr16 - 81132450 81132947 498 browser details YourSeq 101 1674 2262 3000 78.2% chr3 + 111128634 111128796 163 browser details YourSeq 96 1672 2242 3000 93.8% chr1 - 41362425 41430686 68262 browser details YourSeq 89 1666 1775 3000 95.1% chr8 + 59209301 59209466 166 browser details YourSeq 83 1676 1775 3000 94.8% chr10 - 98775277 98775412 136 browser details YourSeq 82 1666 1760 3000 95.7% chr10 - 119502806 119503204 399 browser details YourSeq 77 1672 1762 3000 94.4% chr11 - 98313333 98313537 205 browser details YourSeq 75 1670 1760 3000 97.6% chr4 - 34237238 34237549 312 browser details YourSeq 74 1672 1775 3000 94.0% chr8 + 59902653 59902810 158 browser details YourSeq 74 1665 1775 3000 94.1% chr7 + 141787848 141787965 118 browser details YourSeq 73 1672 1775 3000 96.4% chr2 + 103878845 103879206 362 browser details YourSeq 72 1674 1758 3000 96.3% chr15 + 92205147 92205303 157 browser details YourSeq 69 1674 1756 3000 93.9% chr1 - 112244864 112244960 97 browser details YourSeq 69 1666 1761 3000 93.0% chr1 + 22089217 22089360 144 browser details YourSeq 68 1672 1760 3000 96.0% chr5 - 60610545 60610743 199 browser details YourSeq 68 1787 2244 3000 69.5% chr12 - 47397281 47397418 138 browser details YourSeq 67 1666 1750 3000 94.6% chr7 - 99341851 99341941 91 browser details YourSeq 67 1673 1758 3000 90.5% chr15 + 85563515 85563670 156 browser details YourSeq 67 1674 1760 3000 93.6% chr15 + 72397165 72397303 139

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Gabrg1 gamma-aminobutyric acid (GABA) A receptor, subunit gamma 1 [ Mus musculus (house mouse) ] Gene ID: 14405, updated on 10-Oct-2019

Gene summary

Official Symbol Gabrg1 provided by MGI Official Full Name gamma-aminobutyric acid (GABA) A receptor, subunit gamma 1 provided by MGI Primary source MGI:MGI:103156 See related Ensembl:ENSMUSG00000001260 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as GabaA; GabaA/BZ Expression Biased expression in CNS E18 (RPKM 1.7), frontal lobe adult (RPKM 1.3) and 4 other tissues See more Orthologs human all

Genomic context

Location: 5 C3.1; 5 37.43 cM See Gabrg1 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (70751047..70842617, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (71142286..71233856, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 4 transcripts

Gene: Gabrg1 ENSMUSG00000001260

Description gamma-aminobutyric acid (GABA) A receptor, subunit gamma 1 [Source:MGI Symbol;Acc:MGI:103156] Gene Synonyms GabaA Location Chromosome 5: 70,751,047-70,842,617 reverse strand. GRCm38:CM000998.2 About this gene This gene has 4 transcripts (splice variants), 187 orthologues, 41 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gabrg1-201 ENSMUST00000031119.5 4771 465aa ENSMUSP00000031119.1 Protein coding CCDS19326 Q9R0Y8 TSL:1 GENCODE basic APPRIS P1

Gabrg1-203 ENSMUST00000199705.1 3507 419aa ENSMUSP00000142454.1 Protein coding - A0A0G2JDP9 CDS 5' incomplete TSL:1

Gabrg1-204 ENSMUST00000200126.1 2743 No protein - Retained intron - - TSL:1

Gabrg1-202 ENSMUST00000197068.4 1926 No protein - Retained intron - - TSL:1

111.57 kb Forward strand 70.76Mb 70.78Mb 70.80Mb 70.82Mb 70.84Mb Contigs < AC092716.7 < AC068252.9 Genes (Comprehensive set... < Gabrg1-201protein coding

< Gabrg1-203protein coding

< Gabrg1-202retained intron

< Gabrg1-204retained intron

Regulatory Build

70.76Mb 70.78Mb 70.80Mb 70.82Mb 70.84Mb Reverse strand 111.57 kb

Regulation Legend Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000031119

< Gabrg1-201protein coding

Reverse strand 91.57 kb

ENSMUSP00000031... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... TIGRFAM Neurotransmitter-gated ion-channel Superfamily Neurotransmitter-gated ion-channel ligand-binding domain superfamily

Neurotransmitter-gated ion-channel transmembrane domain superfamily Prints Gamma-aminobutyric-acid A receptor, gamma 1 subunit

Neurotransmitter-gated ion-channel

Gamma-aminobutyric acid A receptor/Glycine receptor alpha

Gamma-aminobutyric-acid A receptor, gamma subunit Pfam Neurotransmitter-gated ion-channel ligand-binding domain Neurotransmitter-gated ion-channel transmembrane domain

PROSITE patterns Neurotransmitter-gated ion-channel, conserved site PANTHER PTHR18945:SF93

Neurotransmitter-gated ion-channel Gene3D Neurotransmitter-gated ion-channel ligand-binding domain superfamily CDD cd19000 cd19054

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 465

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

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