https://www.alphaknockout.com

Mouse Rragb Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Rragb (NCBI Reference Sequence: NM_001004154 ; Ensembl: ENSMUSG00000041658 ) is located on Mouse X. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 11 (Transcript: ENSMUST00000039720). Exon 2~4 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 or hemizygous for a null allele are viable with no gross abnormalities.

Exon 2 starts from about 8.29% of the coding region. Exon 2~4 covers 19.43% of the coding region. The size of effective KO region: ~7172 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 2 3 4 11

Legends Exon of mouse Rragb Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 788 bp section upstream of Exon 2 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 1826 bp section downstream of Exon 4 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.

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

Sequence 12

Summary: Full Length(788bp) | A(30.58% 241) | C(17.39% 137) | T(31.22% 246) | G(20.81% 164)

Note: The 788 bp section upstream of Exon 2 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(1826bp) | A(31.22% 570) | C(13.8% 252) | T(31.87% 582) | G(23.11% 422)

Note: The 1826 bp section downstream of Exon 4 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 788 1 788 788 100.0% chrX + 153140577 153141364 788 browser details YourSeq 36 630 738 788 95.0% chr16 - 34159070 34159484 415 browser details YourSeq 31 707 742 788 85.3% chr12 + 17706419 17706452 34 browser details YourSeq 23 719 741 788 100.0% chr1 - 62685432 62685454 23 browser details YourSeq 23 344 366 788 100.0% chr6 + 17977611 17977633 23 browser details YourSeq 22 722 743 788 100.0% chr18 - 77065430 77065451 22 browser details YourSeq 22 718 744 788 83.4% chr1 + 118889921 118889945 25 browser details YourSeq 21 714 735 788 100.0% chr10 - 39579584 39579606 23 browser details YourSeq 21 720 740 788 100.0% chr10 - 8174543 8174563 21 browser details YourSeq 21 720 740 788 100.0% chr1 - 189390289 189390309 21 browser details YourSeq 21 220 241 788 100.0% chr1 - 26645139 26645161 23 browser details YourSeq 20 722 743 788 95.5% chr1 - 189460221 189460242 22 browser details YourSeq 20 722 741 788 100.0% chr1 - 91424948 91424967 20 browser details YourSeq 20 720 739 788 100.0% chr10 + 91547364 91547383 20 browser details YourSeq 20 408 427 788 100.0% chr1 + 42368098 42368117 20

Note: The 788 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 1826 1 1826 1826 100.0% chrX + 153148537 153150362 1826 browser details YourSeq 36 70 126 1826 90.0% chrX + 49792999 49793054 56 browser details YourSeq 23 993 1018 1826 96.0% chr14 + 80922995 80923022 28 browser details YourSeq 22 64 85 1826 100.0% chr9 + 123334121 123334142 22 browser details YourSeq 22 66 87 1826 100.0% chr3 + 40181670 40181691 22 browser details YourSeq 21 64 84 1826 100.0% chr4 - 42168181 42168201 21 browser details YourSeq 21 772 792 1826 100.0% chr3 - 64903076 64903096 21 browser details YourSeq 21 65 85 1826 100.0% chr19 - 42484711 42484731 21 browser details YourSeq 21 64 84 1826 100.0% chr4_JH584293_random + 7481 7501 21

Note: The 1826 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Rragb Ras-related GTP binding B [ Mus musculus (house mouse) ] Gene ID: 245670, updated on 12-Aug-2019

Gene summary

Official Symbol Rragb provided by MGI Official Full Name Ras-related GTP binding B provided by MGI Primary source MGI:MGI:3038613 See related Ensembl:ENSMUSG00000041658 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 CNS E18 (RPKM 18.2), cerebellum adult (RPKM 16.5) and 9 other tissues See more Orthologs human all

Genomic context

Location: X; X F3 See Rragb in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (153137644..153190192)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (149574501..149606486)

Chromosome X - NC_000086.7

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Transcript information: This gene has 2 transcripts

Gene: Rragb ENSMUSG00000041658

Description Ras-related GTP binding B [Source:MGI Symbol;Acc:MGI:3038613] Location Chromosome X: 153,139,981-153,171,943 forward strand. GRCm38:CM001013.2 About this gene This gene has 2 transcripts (splice variants), 196 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rragb-201 ENSMUST00000039720.10 2258 374aa ENSMUSP00000039013.4 Protein coding CCDS30480 Q6NTA4 TSL:1 GENCODE basic APPRIS P1

Rragb-202 ENSMUST00000144175.2 847 116aa ENSMUSP00000134162.1 Protein coding - G3UYP1 CDS 3' incomplete TSL:5

51.96 kb Forward strand 153.13Mb 153.14Mb 153.15Mb 153.16Mb 153.17Mb 153.18Mb (Comprehensive set... Foxr2-201 >protein coding Rragb-201 >protein coding

Foxr2-202 >protein coding Rragb-202 >protein coding

Contigs AL672293.14 > Regulatory Build

153.13Mb 153.14Mb 153.15Mb 153.16Mb 153.17Mb 153.18Mb Reverse strand 51.96 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

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Transcript: ENSMUST00000039720

31.96 kb Forward strand

Rragb-201 >protein coding

ENSMUSP00000039... MobiDB lite Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase Pfam Gtr1/RagA G protein PANTHER PTHR11259:SF4

Gtr1/RagA G protein Gene3D 3.40.50.300 3.30.450.190

CDD RagA/B

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 374

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

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