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Mouse Rgl2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rgl2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rgl2 gene (NCBI Reference Sequence: NM_009059 ; Ensembl: ENSMUSG00000041354 ) is located on Mouse chromosome 17. 18 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000047503). Exon 3~11 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rgl2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-4F18 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:

Exon 3~11 is not frameshift exon, and covers 51.29% of the coding region. The size of intron 2 for 5'-loxP site insertion: 1284 bp, and the size of intron 11 for 3'-loxP site insertion: 1003 bp. The size of effective cKO region: ~2746 bp. The cKO 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' 10

1 2 3 4 5 6 7 8 9 11 1213 14 15 16 18 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Rgl2 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(9246bp) | A(18.15% 1678) | C(27.98% 2587) | T(24.46% 2262) | G(29.41% 2719)

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.

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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% chr17 + 33928477 33931476 3000 browser details YourSeq 147 1 234 3000 95.7% chr9 + 106845418 106846009 592 browser details YourSeq 132 8 259 3000 88.9% chr11 + 75226674 75226951 278 browser details YourSeq 131 1 216 3000 93.0% chr18 + 75122236 75122769 534 browser details YourSeq 126 1 136 3000 96.4% chr5 - 109898796 109898931 136 browser details YourSeq 126 1 133 3000 97.8% chr17 - 46808661 46808801 141 browser details YourSeq 126 1 133 3000 97.8% chr3 + 28003065 28003204 140 browser details YourSeq 125 1 208 3000 93.2% chr14 + 56819637 56820193 557 browser details YourSeq 125 1 136 3000 96.4% chr14 + 45578687 45578838 152 browser details YourSeq 125 1 223 3000 92.6% chr11 + 87413709 87414276 568 browser details YourSeq 124 1 136 3000 95.6% chr19 - 5908920 5909055 136 browser details YourSeq 124 1 135 3000 96.3% chr16 + 56969203 56969372 170 browser details YourSeq 124 1 133 3000 97.0% chr14 + 82108063 82108414 352 browser details YourSeq 124 1 136 3000 95.6% chr11 + 64460153 64460288 136 browser details YourSeq 124 1 136 3000 95.6% chr10 + 44461572 44461707 136 browser details YourSeq 123 2 136 3000 95.6% chr17 - 33795796 33795930 135 browser details YourSeq 123 1 136 3000 95.6% chr11 - 115740670 115740812 143 browser details YourSeq 123 1 133 3000 96.3% chr11 - 68358718 68358850 133 browser details YourSeq 123 1 133 3000 96.3% chr2 + 37377592 37377724 133 browser details YourSeq 123 1 136 3000 95.6% chr17 + 11825768 11825931 164

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% chr17 + 33934223 33937222 3000 browser details YourSeq 205 1979 2378 3000 91.9% chr12 - 101900696 102019471 118776 browser details YourSeq 166 2106 2345 3000 91.5% chr11 + 87145127 87495381 350255 browser details YourSeq 163 2110 2345 3000 86.3% chr2 + 32780147 32780423 277 browser details YourSeq 151 2106 2566 3000 82.0% chr1 + 180750509 180750787 279 browser details YourSeq 150 2119 2357 3000 94.7% chr16 + 17093367 17093910 544 browser details YourSeq 146 2106 2345 3000 88.5% chr10 - 80595498 80596096 599 browser details YourSeq 143 2195 2384 3000 92.9% chr4 + 123281181 123281531 351 browser details YourSeq 141 2106 2391 3000 86.8% chr12 - 84273064 84273224 161 browser details YourSeq 140 2232 2392 3000 95.0% chr2 + 162961707 162961906 200 browser details YourSeq 139 2117 2349 3000 92.8% chr10 - 93337897 93338277 381 browser details YourSeq 137 2234 2388 3000 94.8% chr13 - 106946076 106946269 194 browser details YourSeq 126 2106 2506 3000 84.4% chr4 - 116422999 116423348 350 browser details YourSeq 126 2106 2379 3000 85.6% chr7 + 124439922 124440084 163 browser details YourSeq 124 2254 2393 3000 94.9% chr12 - 52058612 52058779 168 browser details YourSeq 123 2232 2413 3000 93.7% chr2 + 30272621 30272832 212 browser details YourSeq 121 2106 2373 3000 84.8% chr4 - 143269713 143269853 141 browser details YourSeq 121 2238 2393 3000 94.3% chr6 + 105166835 105167024 190 browser details YourSeq 118 2096 2323 3000 86.8% chr11 + 98521978 98522202 225 browser details YourSeq 118 2234 2367 3000 95.4% chr11 + 20204875 20205038 164

Note: The 3000 bp section downstream of Exon 11 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rgl2 ral guanine nucleotide dissociation stimulator-like 2 [ Mus musculus (house mouse) ] Gene ID: 19732, updated on 12-Aug-2019

Gene summary

Official Symbol Rgl2 provided by MGI Official Full Name ral guanine nucleotide dissociation stimulator-like 2 provided by MGI Primary source MGI:MGI:107483 See related Ensembl:ENSMUSG00000041354 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Rlf; Rgt2; KE1.5; Rab2l Expression Ubiquitous expression in thymus adult (RPKM 47.5), spleen adult (RPKM 34.2) and 28 other tissues See more Orthologs human all

Genomic context

Location: 17; 17 B1 See Rgl2 in Genome Data Viewer

Exon count: 19

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (33929514..33937687)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (34066839..34074632)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 14 transcripts

Gene: Rgl2 ENSMUSG00000041354

Description ral guanine nucleotide dissociation stimulator-like 2 [Source:MGI Symbol;Acc:MGI:107483] Gene Synonyms KE1.5, Rab2l, Rgt2, Rlf Location Chromosome 17: 33,929,543-33,937,687 forward strand. GRCm38:CM001010.2 About this gene This gene has 14 transcripts (splice variants), 100 orthologues, 39 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rgl2- ENSMUST00000047503.15 3252 778aa ENSMUSP00000041082.9 Protein coding CCDS28635 Q61193 TSL:1 201 GENCODE basic APPRIS P1

Rgl2- ENSMUST00000173284.1 943 314aa ENSMUSP00000134312.1 Protein coding - G3UZ21 CDS 5' and 3' 206 incomplete TSL:5

Rgl2- ENSMUST00000234226.1 553 63aa ENSMUSP00000157230.1 Protein coding - A0A3Q4EGY1 CDS 3' incomplete 214

Rgl2- ENSMUST00000173266.7 392 22aa ENSMUSP00000157027.1 Protein coding - A0A3Q4L2R8 CDS 3' incomplete 205 TSL:3

Rgl2- ENSMUST00000234070.1 793 No - Retained - - - 213 protein intron

Rgl2- ENSMUST00000173258.1 788 No - Retained - - TSL:5 204 protein intron

Rgl2- ENSMUST00000173502.1 775 No - Retained - - TSL:3 208 protein intron

Rgl2- ENSMUST00000172468.1 753 No - Retained - - TSL:2 202 protein intron

Rgl2- ENSMUST00000173718.1 720 No - Retained - - TSL:3 209 protein intron

Rgl2- ENSMUST00000173153.7 684 No - Retained - - TSL:2 203 protein intron

Rgl2- ENSMUST00000174442.1 591 No - Retained - - TSL:2 211 protein intron

Rgl2- ENSMUST00000173379.8 569 No - Retained - - TSL:3 207 protein intron

Rgl2- ENSMUST00000174676.1 384 No - Retained - - TSL:2 212 protein intron

Rgl2- ENSMUST00000174410.1 365 No - Retained - - TSL:3 210 protein intron

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28.14 kb Forward strand

33.92Mb 33.93Mb 33.94Mb Genes (Comprehensive set... Tapbp-203 >protein coding Tapbp-204 >retained intronRgl2-213 >retained intron Rgl2-211 >retained intron Wdr46-201 >protein coding

Tapbp-209 >protein coding Rgl2-202 >retained intron Rgl2-206 >protein coding Gm50037-201 >lncRNA

Tapbp-201 >protein coding Rgl2-201 >protein coding Wdr46-203 >retained intron

Tapbp-205 >retained intron Rgl2-205 >protein coding Wdr46-204 >protein coding

Tapbp-206 >protein coding Rgl2-203 >retained intron Wdr46-202 >retained intron

Tapbp-208 >protein coding Rgl2-210 >retained intron

Tapbp-207 >lncRNA Rgl2-214 >protein coding

Tapbp-202 >lncRNA Rgl2-212 >retained intron

Rgl2-207 >retained intron

Rgl2-204 >retained intron

Rgl2-209 >retained intron

Rgl2-208 >retained intron

Contigs CR974462.5 > Genes < Gm19412-201lncRNA < Pfdn6-201protein coding (Comprehensive set...

< Gm19412-202lncRNA < Pfdn6-204protein coding

< Pfdn6-206protein coding

< Pfdn6-202retained intron

< Pfdn6-205protein coding

< Pfdn6-203protein coding

Regulatory Build

33.92Mb 33.93Mb 33.94Mb Reverse strand 28.14 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000047503

8.11 kb Forward strand

Rgl2-201 >protein coding

ENSMUSP00000041... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily Ras guanine nucleotide exchange factor domain superfamily Ubiquitin-like domain superfamily

SMART Ras-like guanine nucleotide exchange factor, N-terminal Ras-associating (RA) domain

Ras guanine-nucleotide exchange factors catalytic domain Pfam Ras guanine-nucleotide exchange factors catalytic domain Ras-associating (RA) domain

Ras-like guanine nucleotide exchange factor, N-terminal PROSITE profiles Ras-like guanine nucleotide exchange factor, N-terminal Ras-associating (RA) domain

Ras guanine-nucleotide exchange factors catalytic domain PROSITE patterns Ras guanine-nucleotide exchange factor, conserved site PANTHER Ral guanine nucleotide dissociation stimulator-like 2

Ras-like guanine nucleotide exchange factor Gene3D 1.20.870.10 Ras guanine-nucleotide exchange factor catalytic domain superfamily 3.10.20.90

CDD Ras-like guanine nucleotide exchange factor, N-terminal cd17211

Ras guanine-nucleotide exchange factors catalytic domain

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

Variant Legend frameshift variant missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 778

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

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