https://www.alphaknockout.com

Mouse Rgs16 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rgs16 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rgs16 gene (NCBI Reference Sequence: NM_011267 ; Ensembl: ENSMUSG00000026475 ) 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: ENSMUST00000027748). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rgs16 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-75F11 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 exhibit increased fatty acid oxidation and circulating ketone levels when fed a high-fat diet. Mice homozygous for a different knock-out allele exhibit impaired Th1 and Th2 chemotaxis and increased susceptibility toparasitic infection.

Exon 3 starts from about 25.87% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 646 bp, and the size of intron 4 for 3'-loxP site insertion: 1504 bp. The size of effective cKO region: ~1010 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 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Rgs16 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(7510bp) | A(23.28% 1748) | C(25.79% 1937) | T(25.85% 1941) | G(25.09% 1884)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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% chr1 + 153738371 153741370 3000 browser details YourSeq 169 70 286 3000 89.8% chr5 + 144084600 144084879 280 browser details YourSeq 160 61 290 3000 90.5% chr7 - 129130412 129130712 301 browser details YourSeq 154 18 218 3000 91.5% chr8 + 77125026 77272600 147575 browser details YourSeq 154 32 290 3000 90.3% chr12 + 100680028 100680284 257 browser details YourSeq 154 32 247 3000 85.5% chr10 + 78083540 78083746 207 browser details YourSeq 151 32 310 3000 89.2% chr1 - 120288583 120288869 287 browser details YourSeq 150 61 279 3000 86.7% chrX + 95674842 95675043 202 browser details YourSeq 149 91 277 3000 93.3% chr10 - 91263654 91263851 198 browser details YourSeq 149 61 346 3000 90.0% chrX + 153218530 153219032 503 browser details YourSeq 148 61 244 3000 91.4% chr7 - 123441304 123646368 205065 browser details YourSeq 148 55 250 3000 90.3% chr10 - 6008641 6008903 263 browser details YourSeq 148 61 310 3000 83.1% chr1 + 161206800 161207017 218 browser details YourSeq 147 65 310 3000 87.8% chr11 + 106358349 106358610 262 browser details YourSeq 145 61 284 3000 83.5% chr9 + 94632660 94632863 204 browser details YourSeq 145 63 273 3000 88.7% chr9 + 75956686 75957095 410 browser details YourSeq 144 61 242 3000 92.5% chr12 - 91520068 91520249 182 browser details YourSeq 143 32 310 3000 83.1% chr10 + 23891263 23891516 254 browser details YourSeq 143 60 236 3000 91.0% chr10 + 18644464 18644641 178 browser details YourSeq 142 88 348 3000 84.1% chr13 - 93511673 93511922 250

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 + 153742381 153745380 3000 browser details YourSeq 73 855 1105 3000 77.3% chr16 - 13220994 13221165 172 browser details YourSeq 62 109 552 3000 77.2% chr13 + 53336467 53336821 355 browser details YourSeq 60 855 1080 3000 79.8% chr16 - 36003851 36004046 196 browser details YourSeq 60 856 981 3000 77.7% chr1 + 98072860 98072964 105 browser details YourSeq 58 868 993 3000 81.3% chr12 - 17519658 17519763 106 browser details YourSeq 51 855 1090 3000 72.6% chr12 - 16615019 16615197 179 browser details YourSeq 51 856 968 3000 79.0% chr12 - 3863649 3863741 93 browser details YourSeq 50 676 894 3000 91.6% chr17 - 7649479 7649887 409 browser details YourSeq 50 863 993 3000 93.2% chr13 - 98826687 98826833 147 browser details YourSeq 49 856 1087 3000 74.6% chr19 - 7390874 7391060 187 browser details YourSeq 48 863 981 3000 73.2% chr13 - 26433626 26433726 101 browser details YourSeq 47 855 1096 3000 92.8% chr13 - 53388866 53389401 536 browser details YourSeq 46 855 965 3000 76.0% chr1 + 175545436 175545526 91 browser details YourSeq 46 1095 1363 3000 67.5% chr1 + 169691840 169692288 449 browser details YourSeq 45 867 985 3000 74.6% chr10 - 41112590 41112687 98 browser details YourSeq 45 855 902 3000 98.0% chr14 + 61419795 61419848 54 browser details YourSeq 44 856 967 3000 72.0% chr16 + 96045432 96045508 77 browser details YourSeq 44 856 1059 3000 89.1% chr1 + 192514371 192514765 395 browser details YourSeq 43 862 967 3000 75.6% chr12 - 82540386 82540471 86

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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

Gene and protein information: Rgs16 regulator of G-protein signaling 16 [ Mus musculus (house mouse) ] Gene ID: 19734, updated on 12-Aug-2019

Gene summary

Official Symbol Rgs16 provided by MGI Official Full Name regulator of G-protein signaling 16 provided by MGI Primary source MGI:MGI:108407 See related Ensembl:ENSMUSG00000026475 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 Rgsr; Rgs14 Expression Broad expression in liver E18 (RPKM 12.8), CNS E11.5 (RPKM 10.4) and 15 other tissues See more Orthologs human all

Genomic context

Location: 1 G3; 1 65.43 cM See Rgs16 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (153740353..153745468)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (155587483..155592598)

Chromosome 1 - NC_000067.6

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

Transcript information: This gene has 1 transcript

Gene: Rgs16 ENSMUSG00000026475

Description regulator of G-protein signaling 16 [Source:MGI Symbol;Acc:MGI:108407] Gene Synonyms Rgsr Location Chromosome 1: 153,740,349-153,745,468 forward strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 161 orthologues, 23 paralogues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rgs16-201 ENSMUST00000027748.7 2339 201aa ENSMUSP00000027748.7 Protein coding CCDS15376 P97428 Q542U0 TSL:1 GENCODE basic APPRIS P1

25.12 kb Forward strand 153.735Mb 153.740Mb 153.745Mb 153.750Mb 153.755Mb Genes (Comprehensive set... Rgs16-201 >protein coding Rnasel-201 >protein coding

Rnasel-205 >protein coding

Gm28512-201 >lncRNA

Rnasel-206 >protein coding

Rnasel-203 >retained intron

Rnasel-204 >protein coding

Contigs AC154140.7 > Genes < C230024C17Rik-201lncRNA < Gm28513-201lncRNA (Comprehensive set...

Regulatory Build

153.735Mb 153.740Mb 153.745Mb 153.750Mb 153.755Mb Reverse strand 25.12 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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

Transcript: ENSMUST00000027748

5.12 kb Forward strand

Rgs16-201 >protein coding

ENSMUSP00000027... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily RGS domain superfamily SMART RGS domain Prints RGS domain Pfam RGS domain PROSITE profiles RGS domain PANTHER PTHR10845:SF187

PTHR10845 Gene3D RGS, subdomain 1/3

1.10.167.10 CDD cd08710

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 201

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

Page 7 of 7