https://www.alphaknockout.com

Mouse Gabrr1 Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Gabrr1 (NCBI Reference Sequence: NM_008075 ; Ensembl: ENSMUSG00000028280 ) is located on Mouse 4. 10 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000029947). Exon 3~7 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO 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 3 starts from about 12.29% of the coding region. Exon 3~7 covers 43.26% of the coding region. The size of effective KO region: ~9257 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 3 4 5 6 7 10

Legends Exon of mouse Gabrr1 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1930 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1939 bp section downstream of Exon 7 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(1930bp) | A(26.63% 514) | C(21.55% 416) | T(22.85% 441) | G(28.96% 559)

Note: The 1930 bp section upstream of Exon 3 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(1939bp) | A(25.17% 488) | C(21.71% 421) | T(29.24% 567) | G(23.88% 463)

Note: The 1939 bp section downstream of Exon 7 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 1930 1 1930 1930 100.0% chr4 + 33146992 33148921 1930 browser details YourSeq 75 975 1078 1930 91.2% chr1 + 91479094 91479197 104 browser details YourSeq 71 968 1210 1930 77.6% chr5 + 33002461 33002665 205 browser details YourSeq 68 984 1076 1930 89.3% chr7 - 30800824 30800916 93 browser details YourSeq 67 968 1074 1930 92.4% chr10 + 117449469 117707480 258012 browser details YourSeq 64 968 1079 1930 82.2% chr7 - 24469331 24469441 111 browser details YourSeq 63 961 1063 1930 88.8% chr12 + 53387010 53387112 103 browser details YourSeq 60 964 1209 1930 63.7% chr1 - 87816656 87816854 199 browser details YourSeq 59 969 1072 1930 92.8% chr4 - 84210140 84210247 108 browser details YourSeq 59 968 1131 1930 96.9% chr3 - 66681823 66931716 249894 browser details YourSeq 58 970 1204 1930 66.7% chr11 - 11194188 11194379 192 browser details YourSeq 58 968 1061 1930 88.2% chr10 - 120270988 120271091 104 browser details YourSeq 58 971 1058 1930 83.8% chr5 + 67490819 67490907 89 browser details YourSeq 55 957 1055 1930 79.6% chr5 - 126351144 126351249 106 browser details YourSeq 55 997 1076 1930 85.8% chr11 + 85273460 85273540 81 browser details YourSeq 54 983 1088 1930 84.9% chrX + 94314739 94314841 103 browser details YourSeq 52 982 1053 1930 86.2% chr13 - 93798486 93798557 72 browser details YourSeq 52 969 1049 1930 88.9% chr1 - 182356324 182356403 80 browser details YourSeq 52 1237 1307 1930 92.1% chr10 + 111046510 111046604 95 browser details YourSeq 51 996 1064 1930 83.9% chr1 - 166890280 166890345 66

Note: The 1930 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 1939 1 1939 1939 100.0% chr4 + 33158179 33160117 1939 browser details YourSeq 50 1292 1846 1939 66.1% chr13 - 37432353 37432701 349 browser details YourSeq 43 1497 1649 1939 94.2% chr2 - 137885639 137886046 408 browser details YourSeq 36 1296 1337 1939 92.9% chr12 + 75746355 75746396 42 browser details YourSeq 34 1291 1350 1939 77.0% chr19 + 22955695 22955752 58 browser details YourSeq 33 1290 1336 1939 85.2% chr16 - 33115855 33115901 47 browser details YourSeq 33 1291 1338 1939 85.4% chr10 + 119551645 119551691 47 browser details YourSeq 32 1291 1334 1939 86.4% chr16 + 23217946 23217989 44 browser details YourSeq 31 1298 1330 1939 97.0% chr1 - 36394893 36394925 33 browser details YourSeq 30 1290 1332 1939 89.5% chr15 - 55348844 55348887 44 browser details YourSeq 30 1500 1533 1939 97.0% chr1 + 9614888 9614922 35 browser details YourSeq 28 1461 1508 1939 96.8% chr17 - 28346155 28346202 48 browser details YourSeq 28 1310 1337 1939 100.0% chr13 - 24533560 24533587 28 browser details YourSeq 27 1495 1523 1939 100.0% chr2 - 45741123 45741173 51 browser details YourSeq 26 1282 1317 1939 75.8% chr3 - 130889726 130889758 33 browser details YourSeq 26 1550 1584 1939 96.5% chr17 - 4178312 4178347 36 browser details YourSeq 26 245 274 1939 96.7% chr14 - 46276263 46276296 34 browser details YourSeq 26 1499 1531 1939 83.4% chr1 + 71625609 71625639 31 browser details YourSeq 25 1550 1577 1939 96.3% chr2 - 156471834 156471862 29 browser details YourSeq 25 1291 1333 1939 79.1% chr2 - 71109010 71109052 43

Note: The 1939 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.

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Gene and 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

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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 (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

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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/ 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.

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