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Mouse Rgs18 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rgs18 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rgs18 gene ( NCBI Reference Sequence: NM_022881 ; Ensembl: ENSMUSG00000026357 ) is located on mouse chromosome 1. 5 exons are identified , with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript Rgs18- 201: ENSMUST00000027603). 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 Rgs18 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-340I20 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: Homozygotes for a null allele show reduced thermal nociception threshold, increased startle reflex, thrombocytopenia, defective megakaryopoiesis, and increased platelet aggregation. Homozygotes for a different null allele show decreased bleeding time, increased platelet aggregation, and thrombosis.

The knockout of Exon 3 will result in frameshift of the gene, and covers 8.79% of the coding region. The size of intron 2 for 5'-loxP site insertion: 984 bp, and the size of intron 3 for 3'-loxP site insertion: 17431 bp.

The size of effective cKO region: ~1061 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 protein 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 2 3 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Rgs18 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(7062bp) | A(33.18% 2343) | C(15.59% 1101) | G(16.79% 1186) | T(34.44% 2432)

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% chr1 - 144773833 144776832 3000 browser details YourSeq 144 30 314 3000 88.9% chr9 + 32248885 32249423 539 browser details YourSeq 133 30 314 3000 87.8% chr17 + 94478859 94479395 537 browser details YourSeq 119 27 314 3000 87.5% chr18 - 46147827 46148381 555 browser details YourSeq 115 70 314 3000 84.6% chrX + 23638792 23639308 517 browser details YourSeq 113 39 337 3000 84.8% chr14 - 102857210 102857795 586 browser details YourSeq 112 32 285 3000 90.0% chr15 - 6633775 6634327 553 browser details YourSeq 110 27 288 3000 80.8% chr1 - 84978393 84978890 498 browser details YourSeq 110 27 188 3000 85.9% chr2 + 77711535 77711697 163 browser details YourSeq 110 27 192 3000 87.3% chr12 + 78984583 78984753 171 browser details YourSeq 109 27 279 3000 84.8% chr7 - 112435901 112436434 534 browser details YourSeq 107 39 188 3000 89.1% chr6 - 33549522 33549671 150 browser details YourSeq 106 84 288 3000 83.6% chr1_GL456221_random - 11876 12325 450 What is ch rom_random? browser details YourSeq 103 27 188 3000 87.6% chr11 - 35757696 35757860 165 browser details YourSeq 102 27 192 3000 88.1% chrX + 157866915 157867084 170 browser details YourSeq 100 44 188 3000 84.9% chr8 + 47344160 47344305 146 browser details YourSeq 99 49 187 3000 86.3% chr1 + 72153793 72153932 140 browser details YourSeq 98 29 184 3000 86.7% chr13 - 93271425 93271582 158 browser details YourSeq 98 39 279 3000 83.0% chr8 + 34482617 34483100 484 browser details YourSeq 98 27 188 3000 80.7% chr19 + 60175710 60175874 165

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% chr1 - 144770271 144773270 3000 browser details YourSeq 172 191 424 3000 90.2% chr2 + 117672825 117673058 234 browser details YourSeq 171 192 408 3000 91.8% chrX - 41516037 41516256 220 browser details YourSeq 167 191 430 3000 89.0% chr4 - 94833042 94833306 265 browser details YourSeq 166 191 408 3000 90.3% chr4 + 116375971 116376193 223 browser details YourSeq 165 198 425 3000 89.1% chr13 - 63785692 63785929 238 browser details YourSeq 164 192 421 3000 90.3% chr3 - 58307660 58307901 242 browser details YourSeq 164 191 415 3000 87.1% chrX + 73902079 73902301 223 browser details YourSeq 164 192 408 3000 91.1% chr2 + 155325121 155325341 221 browser details YourSeq 163 191 408 3000 89.9% chr11 - 18250771 18250990 220 browser details YourSeq 163 192 409 3000 87.6% chr12 + 91991306 92151172 159867 browser details YourSeq 161 192 416 3000 87.4% chr9 - 86102175 86102400 226 browser details YourSeq 161 197 416 3000 89.7% chr3 + 146668861 146669087 227 browser details YourSeq 159 197 421 3000 92.5% chr14 - 73169068 73169295 228 browser details YourSeq 159 191 407 3000 88.4% chr7 + 102945204 102945418 215 browser details YourSeq 158 191 424 3000 89.8% chr14 + 106496398 106496628 231 browser details YourSeq 157 204 424 3000 89.5% chrX - 60193802 60194026 225 browser details YourSeq 157 192 424 3000 88.5% chr19 - 23860453 23860831 379 browser details YourSeq 157 197 408 3000 89.9% chr12 - 113965204 113965420 217 browser details YourSeq 157 191 424 3000 85.8% chr2 + 39801068 39801299 232

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://beta.alphaknockout.cyagen.net Gene and protein information: Rgs18 regulator of G-protein signaling 18 [ Mus musculus (house mouse) ] Gene ID: 64214, updated on 12-Aug-2019

Gene summary

Official Symbol Rgs18 provided by MGI Official Full Name regulator of G-protein signaling 18 provided by MGI Primary source MGI:MGI:1927498 See related Ensembl:ENSMUSG00000026357 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 Expression Biased expression in liver E14 (RPKM 3.7), liver E18 (RPKM 2.8) and 7 other tissues See more Orthologs human all

Genomic context

Location: 1 F; 1 62.99 cM See Rgs18 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (144752683..144775427, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (146599971..146622551, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 1 transcript

Gene: Rgs18 ENSMUSG00000026357

Description regulator of G-protein signaling 18 [Source:MGI Symbol;Acc:MGI:1927498] Location Chromosome 1: 144,752,683-144,775,435 reverse strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 128 orthologues, 23 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rgs18-201 ENSMUST00000027603.3 2031 235aa ENSMUSP00000027603.3 Protein coding CCDS15350 Q544K2 Q99PG4 TSL:1 GENCODE basic APPRIS P1

42.75 kb Forward strand

144.62Mb 144.63Mb 144.64Mb 144.65Mb 144.66Mb Contigs < AC102163.14 Genes (Comprehensive set from GENCODE M... < Rgs18-201protein coding

Regulatory Build

144.62Mb 144.63Mb 144.64Mb 144.65Mb 144.66Mb Reverse strand 42.75 kb

Gene Legend Protein Coding

merged Ensembl/Havana

Regulation Legend

Open Chromatin Promoter Promoter Flank

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Transcript: ENSMUST00000027603

< Rgs18-201protein coding

Reverse strand 22.75 kb

ENSMUSP000000276... Superfamily RGS domain superfamily SMART RGS domain Prints RGS domain Pfam RGS domain PROSITE profiles RGS domain PANTHER PTHR10845

Regulator of G-protein signalling 18

Gene3D RGS, subdomain 1/3

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 200 235

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

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