http://www.alphaknockout.com/ Mouse Gabra3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gabra3 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gabra3 ( NCBI Reference Sequence: NM_008067 ; Ensembl: ENSMUSG00000031343 ) is located on mouse X. 10 are identified , with the ATG start codon in 2 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000055966). 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 Gabra3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-355F20 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: Hemizygous males exhibit hyperactivity and an increased startle response with impaired prepulse inhibition.

The knockout of Exon 3 will result in frameshift of the gene, and covers 8.27% of the coding region. The size of intron 2 for 5'-loxP site insertion: 12492 bp, and the size of intron 3 for 3'-loxP site insertion: 28690 bp. The size of effective cKO region: ~955 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and translation cannot be predicted at existing technological level.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Gabra3 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7122bp) | A(30.48% 2171) | C(17.45% 1243) | G(17.75% 1264) | T(34.32% 2444)

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% chrX - 72540435 72543434 3000 browser details YourSeq 358 2147 2579 3000 94.3% chr5 + 45241902 45252438 10537 browser details YourSeq 358 2146 2543 3000 94.7% chr15 + 74212886 74213281 396 browser details YourSeq 357 2147 2543 3000 95.0% chr17 - 23908357 23908752 396 browser details YourSeq 353 2147 2543 3000 94.1% chr3 + 55102140 55102527 388 browser details YourSeq 350 2142 2543 3000 93.6% chr18 - 14042153 14042545 393 browser details YourSeq 349 2164 2549 3000 95.6% chr9 - 25034034 25034434 401 browser details YourSeq 347 2130 2548 3000 94.2% chr13 - 8923194 8923633 440 browser details YourSeq 347 2164 2581 3000 94.9% chr9 + 122483106 122483687 582 browser details YourSeq 344 2151 2536 3000 94.9% chr13 + 10986050 10986444 395 browser details YourSeq 342 2147 2552 3000 94.1% chr6 - 87949908 87950322 415 browser details YourSeq 342 2147 2543 3000 93.3% chrX + 18309883 18310275 393 browser details YourSeq 341 2147 2543 3000 94.4% chr9 - 25031767 25032177 411 browser details YourSeq 341 2164 2548 3000 94.2% chr18 + 73891620 73892002 383 browser details YourSeq 340 2147 2536 3000 93.6% chr2 - 168408990 168409368 379 browser details YourSeq 340 2146 2548 3000 93.6% chr18 + 16070608 16073301 2694 browser details YourSeq 338 2158 2543 3000 94.3% chr12 + 3184497 3184887 391 browser details YourSeq 337 2153 2543 3000 93.8% chr11 - 113527829 113528227 399 browser details YourSeq 337 2163 2543 3000 93.9% chr12 + 11000058 11000434 377 browser details YourSeq 337 2158 2549 3000 93.2% chr12 + 10997762 10998149 388

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% chrX - 72536813 72539812 3000 browser details YourSeq 62 1774 2065 3000 75.4% chr12 - 13429632 13429751 120 browser details YourSeq 61 1725 1821 3000 95.6% chr2 - 97892442 97892922 481 browser details YourSeq 60 1772 2015 3000 72.8% chr4 - 78829020 78829091 72 browser details YourSeq 59 1782 2116 3000 95.4% chr10 - 25516750 25517086 337 browser details YourSeq 58 1766 1836 3000 84.2% chr12 + 42613415 42613477 63 browser details YourSeq 57 1766 1822 3000 100.0% chr6 + 35092247 35092303 57 browser details YourSeq 56 1765 1822 3000 98.3% chr12 - 75490885 75490942 58 browser details YourSeq 56 1766 1822 3000 100.0% chr8 + 42991480 42991538 59 browser details YourSeq 56 1766 1823 3000 98.3% chr3 + 63682174 63682231 58 browser details YourSeq 55 1766 1822 3000 98.3% chrX - 14448461 14448517 57 browser details YourSeq 55 1766 1822 3000 98.3% chr5 - 75787473 75787529 57 browser details YourSeq 55 1766 1822 3000 98.3% chr3 - 127296621 127296677 57 browser details YourSeq 55 1766 1822 3000 98.3% chr17 - 16171913 16171969 57 browser details YourSeq 55 1766 1822 3000 98.3% chr14 - 24590757 24590813 57 browser details YourSeq 55 1766 1822 3000 98.3% chr14 - 8581806 8581862 57 browser details YourSeq 55 1766 1822 3000 98.3% chr13 - 49109141 49109197 57 browser details YourSeq 55 1766 1822 3000 98.3% chr11 - 57447496 57447552 57 browser details YourSeq 55 1766 1820 3000 100.0% chrX + 52707929 52707983 55 browser details YourSeq 55 1766 1822 3000 98.3% chr3 + 18580585 18580641 57

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 http://www.alphaknockout.com/ Gene and protein information: Gabra3 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 3 [ Mus musculus (house mouse) ] Gene ID: 14396, updated on 13-Dec-2020

Gene summary

Official Symbol Gabra3 provided by MGI Official Full Name gamma-aminobutyric acid (GABA) A receptor, subunit alpha 3 provided by MGI Primary source MGI:MGI:95615 See related Ensembl:ENSMUSG00000031343 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 Gabr; Gabra-3 Expression Biased expression in frontal lobe adult (RPKM 17.8), cortex adult (RPKM 14.7) and 10 other tissues See more Orthologs human all NEW Try the new Gene table Try the new Transcript table

Genomic context

Location: X A7.3; X 37.24 cM See Gabra3 in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) X NC_000086.8 (71476282..71700525, complement)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (72432676..72656919, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (69678015..69901585, complement)

Chromosome X - NC_000086.8

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Transcript information: This gene has 3 transcripts

Gene: Gabra3 ENSMUSG00000031343

Description gamma-aminobutyric acid (GABA) A receptor, subunit alpha 3 [Source:MGI Symbol;Acc:MGI:95615] Gene Synonyms Gabra-3 Location Chromosome X: 71,476,287-71,700,454 reverse strand. GRCm39:CM001013.3 About this gene This gene has 3 transcripts (splice variants), 196 orthologues, 41 paralogues and is associated with 4 phenotypes. Transcripts

UniProt Name Transcript ID bp Protein Translation ID Biotype CCDS Flags Match

Gabra3- ENSMUST00000055966.13 3662 492aa ENSMUSP00000062638.7 Protein coding CCDS30187 P26049 TSL:1 201 GENCODE basic APPRIS P2

Gabra3- ENSMUST00000114554.3 1790 533aa ENSMUSP00000110201.3 Protein coding - B1AVY2 TSL:5 202 GENCODE basic APPRIS ALT2

Gabra3- ENSMUST00000137517.2 589 No - Processed - - TSL:3 203 protein transcript

244.17 kb Forward strand 71.5Mb 71.6Mb 71.7Mb Gm14735-201 >processed pseudogene AL731695.2-201 >snRNA (Comprehensive set...

AL731695.1-201 >snoRNA

Contigs BX119994.7 > AL731695.11 >

Genes (Comprehensive set... < Gabra3-201protein coding

< Gabra3-202protein coding

< Gabra3-203processed transcript

< Mir767-201miRNA

< Mir105-201miRNA

Regulatory Build

71.5Mb 71.6Mb 71.7Mb Reverse strand 244.17 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene pseudogene processed transcript

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Transcript: ENSMUST00000055966

< Gabra3-201protein coding

Reverse strand 223.51 kb

ENSMUSP00000062... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... TIGRFAM Neurotransmitter-gated ion-channel Superfamily Neurotransmitter-gated ion-channel transmembrane domain superfamily

Neurotransmitter-gated ion-channel ligand-binding domain superfamily Prints Neurotransmitter-gated ion-channel

Gamma-aminobutyric-acid A receptor, alpha subunit

Gamma-aminobutyric acid A receptor/ alpha

Gamma-aminobutyric-acid A receptor, alpha 3 subunit Pfam Neurotransmitter-gated ion-channel ligand-binding domain

Neurotransmitter-gated ion-channel transmembrane domain PROSITE patterns Neurotransmitter-gated ion-channel, conserved site PANTHER Neurotransmitter-gated ion-channel

PTHR18945:SF216 Gene3D Neurotransmitter-gated ion-channel ligand-binding domain superfamily CDD cd19036 cd19052

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 492

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

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