https://www.alphaknockout.com

Mouse Gabrr1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gabrr1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gabrr1 gene (NCBI Reference Sequence: NM_008075 ; Ensembl: ENSMUSG00000028280 ) is located on Mouse chromosome 4. 10 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000029947). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Gabrr1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-122L3 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: Mice homozygous for a knock-out allele display altered visual processing in the retina. Mice homozygous for a different knock-out allele exhibit alterations of mechanical pain sensitivity, GABA-inhibited spinal cord responses, and olfactory function.

Exon 2 starts from about 8.54% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 14161 bp, and the size of intron 3 for 3'-loxP site insertion: 2341 bp. The size of effective cKO region: ~2591 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Gabrr1 Homology arm cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

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(9091bp) | A(24.88% 2262) | C(21.99% 1999) | T(25.79% 2345) | G(27.33% 2485)

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.

Page 3 of 7 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr4 + 33143688 33146687 3000 browser details YourSeq 129 2558 2988 3000 79.2% chr11 + 110541766 110542045 280 browser details YourSeq 117 2566 2948 3000 83.1% chr13 - 51558836 51559166 331 browser details YourSeq 115 5 450 3000 77.0% chr13 + 24875502 24875717 216 browser details YourSeq 113 2611 2965 3000 91.8% chr1 + 185558651 185559134 484 browser details YourSeq 107 2561 2948 3000 89.6% chr15 - 95679092 95679586 495 browser details YourSeq 107 2676 2942 3000 83.0% chr10 + 97165262 97165483 222 browser details YourSeq 101 302 497 3000 85.4% chr6 + 52656601 52656796 196 browser details YourSeq 96 2559 2988 3000 76.7% chr17 - 85509305 85509542 238 browser details YourSeq 96 49 453 3000 76.7% chr6 + 82959105 82959292 188 browser details YourSeq 96 2305 2944 3000 75.9% chr1 + 75259164 75259455 292 browser details YourSeq 95 2779 2991 3000 88.0% chr5 - 113498937 113499346 410 browser details YourSeq 92 2780 2969 3000 89.6% chr11 + 95207666 95207888 223 browser details YourSeq 90 2607 2970 3000 90.9% chr17 - 31190028 31190688 661 browser details YourSeq 89 321 479 3000 87.0% chr11 - 44065285 44076357 11073 browser details YourSeq 89 314 496 3000 81.4% chr18 + 4628615 4628788 174 browser details YourSeq 87 293 481 3000 81.0% chr12 - 52072566 52072752 187 browser details YourSeq 86 2758 2969 3000 88.9% chr18 - 82374787 82375113 327 browser details YourSeq 85 317 478 3000 84.0% chr4 - 148123800 148123961 162 browser details YourSeq 84 2633 2938 3000 73.9% chr19 - 54428904 54429060 157

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% chr4 + 33149279 33152278 3000 browser details YourSeq 165 1806 2039 3000 87.9% chr7 + 24812513 24813109 597 browser details YourSeq 152 1805 2025 3000 90.0% chr2 + 180231746 180232163 418 browser details YourSeq 141 1808 2022 3000 86.7% chr15 + 57152485 57616357 463873 browser details YourSeq 139 1684 1969 3000 85.8% chr5 + 132740139 132740464 326 browser details YourSeq 136 1821 2025 3000 86.5% chrX - 101045529 101045906 378 browser details YourSeq 134 1800 1982 3000 86.9% chr7 - 73719443 73719632 190 browser details YourSeq 134 1835 2009 3000 91.0% chr11 + 60010760 60011089 330 browser details YourSeq 129 1799 1985 3000 85.6% chrX - 164380295 164380481 187 browser details YourSeq 128 1808 1985 3000 87.5% chr7 + 113365277 113365455 179 browser details YourSeq 126 1833 2017 3000 87.5% chr2 + 132651386 132651734 349 browser details YourSeq 123 1800 1981 3000 83.1% chr19 + 59514247 59514426 180 browser details YourSeq 122 1800 1982 3000 81.0% chr12 - 116049562 116049734 173 browser details YourSeq 120 1800 1982 3000 86.2% chr14 + 48363712 48363896 185 browser details YourSeq 119 1800 2006 3000 86.5% chr1 - 127103300 127103672 373 browser details YourSeq 117 1800 1972 3000 84.4% chr8 + 112213321 112213494 174 browser details YourSeq 117 1837 2022 3000 88.3% chr2 + 120305812 120306375 564 browser details YourSeq 116 1833 1982 3000 89.5% chr10 + 98674681 98674829 149 browser details YourSeq 115 1800 1972 3000 81.3% chr8 - 84455509 84455679 171 browser details YourSeq 114 1837 2037 3000 87.1% chr10 + 87374109 87374465 357

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

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Gabrr1 gamma-aminobutyric acid (GABA) C receptor, subunit rho 1 [ Mus musculus (house mouse) ] Gene ID: 14408, updated on 22-Oct-2019

Gene summary

Official Symbol Gabrr1 provided by MGI Official Full Name gamma-aminobutyric acid (GABA) C receptor, subunit rho 1 provided by MGI Primary source MGI:MGI:95625 See related Ensembl:ENSMUSG00000028280 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 GABA-C Expression Biased expression in genital fat pad adult (RPKM 2.0) and CNS E18 (RPKM 0.1) See more Orthologs human all

Genomic context

Location: 4 A5; 4 14.68 cM See Gabrr1 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (33132556..33163588)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (33219531..33250563)

Chromosome 4 - NC_000070.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 1 transcript

Gene: Gabrr1 ENSMUSG00000028280

Description gamma-aminobutyric acid (GABA) C receptor, subunit rho 1 [Source:MGI Symbol;Acc:MGI:95625] Gene Synonyms GABA-C Location Chromosome 4: 33,132,521-33,163,588 forward strand. GRCm38:CM000997.2 About this gene This gene has 1 transcript (splice variant), 224 orthologues, 41 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gabrr1-201 ENSMUST00000029947.5 2286 480aa ENSMUSP00000029947.6 Protein coding CCDS18021 P56475 TSL:1 GENCODE basic APPRIS P1

51.07 kb Forward strand

33.13Mb 33.14Mb 33.15Mb 33.16Mb 33.17Mb Genes (Comprehensive set... Gabrr1-201 >protein coding

Contigs < AL670464.12 Regulatory Build

33.13Mb 33.14Mb 33.15Mb 33.16Mb 33.17Mb Reverse strand 51.07 kb

Regulation Legend

CTCF Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000029947

31.07 kb Forward strand

Gabrr1-201 >protein coding

ENSMUSP00000029... Transmembrane heli... MobiDB lite 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, Rho1

Gamma-aminobutyric acid A receptor/Glycine receptor alpha

Gamma-aminobutyric-acid A receptor, Rho

Neurotransmitter-gated ion-channel Pfam Neurotransmitter-gated ion-channel ligand-binding domain

Neurotransmitter-gated ion-channel transmembrane domain PROSITE patterns Neurotransmitter-gated ion-channel, conserved site PANTHER PTHR18945:SF30

Neurotransmitter-gated ion-channel Gene3D Neurotransmitter-gated ion-channel ligand-binding domain superfamily CDD cd19005 cd19059

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

Variant Legend synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480

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

Page 7 of 7