https://www.alphaknockout.com

Mouse Rit2 Knockout Project (CRISPR/Cas9)

Objective: To create a Rit2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rit2 gene (NCBI Reference Sequence: NM_009065 ; Ensembl: ENSMUSG00000057455 ) is located on Mouse chromosome 18. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000153060). Exon 3 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:

Exon 3 starts from about 24.73% of the coding region. Exon 3 covers 11.37% of the coding region. The size of effective KO region: ~74 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 5

Legends Exon of mouse Rit2 Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 2000 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 2000 bp section downstream 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(24.8% 496) | C(19.5% 390) | T(33.35% 667) | G(22.35% 447)

Note: The 2000 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(2000bp) | A(34.75% 695) | C(17.55% 351) | T(29.25% 585) | G(18.45% 369)

Note: The 2000 bp section downstream 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr18 - 31212732 31214731 2000 browser details YourSeq 416 11 695 2000 85.8% chrX + 100551804 100552461 658 browser details YourSeq 389 32 642 2000 83.9% chr17 - 53352634 53353251 618 browser details YourSeq 383 4 675 2000 87.5% chr7 - 120023784 120024515 732 browser details YourSeq 372 42 642 2000 83.0% chr10 + 130520441 130521040 600 browser details YourSeq 357 32 595 2000 88.6% chr13 + 59047088 59047656 569 browser details YourSeq 352 4 600 2000 85.3% chr13 - 46307734 46308326 593 browser details YourSeq 350 87 721 2000 82.0% chr8 - 69013714 69014342 629 browser details YourSeq 343 1 674 2000 87.0% chr17 - 42721766 42722357 592 browser details YourSeq 341 32 642 2000 90.0% chrX - 75276607 75277360 754 browser details YourSeq 330 4 695 2000 84.2% chr13 + 100368921 100369523 603 browser details YourSeq 328 4 492 2000 86.4% chr8 - 106456815 106457292 478 browser details YourSeq 327 4 677 2000 85.1% chr8 + 46134630 46135213 584 browser details YourSeq 326 4 638 2000 86.4% chr4 - 11810669 11811306 638 browser details YourSeq 325 4 677 2000 86.7% chr7 - 62970328 62970894 567 browser details YourSeq 321 6 679 2000 86.2% chr2 - 41353919 41354390 472 browser details YourSeq 317 4 475 2000 88.1% chr19 - 34801045 34801606 562 browser details YourSeq 314 32 556 2000 85.3% chr18 + 10283355 10283989 635 browser details YourSeq 311 4 642 2000 85.4% chr9 - 121199188 121199703 516 browser details YourSeq 309 4 695 2000 85.4% chr14 + 14728160 14728739 580

Note: The 2000 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 2000 1 2000 2000 100.0% chr18 - 31210658 31212657 2000 browser details YourSeq 284 1401 1726 2000 93.9% chrX - 94336557 94336885 329 browser details YourSeq 279 1401 1720 2000 94.9% chr4 - 153122646 153122971 326 browser details YourSeq 278 1401 1722 2000 93.5% chr1 + 90438430 90438756 327 browser details YourSeq 276 1401 1721 2000 94.6% chr18 - 60588888 60589212 325 browser details YourSeq 274 1401 1722 2000 93.7% chr4 - 150732612 150732940 329 browser details YourSeq 273 1401 1720 2000 93.7% chr16 + 91988075 91988400 326 browser details YourSeq 273 1401 1720 2000 94.0% chr10 + 127735721 127736046 326 browser details YourSeq 272 1401 1721 2000 93.4% chr15 - 84361685 84362011 327 browser details YourSeq 269 1401 1720 2000 93.9% chr13 - 51376306 51376633 328 browser details YourSeq 269 1401 1720 2000 93.3% chr2 + 72844771 72845096 326 browser details YourSeq 269 1405 1720 2000 93.1% chr10 + 122892212 122892535 324 browser details YourSeq 267 1400 1720 2000 94.7% chr9 - 40926752 40927076 325 browser details YourSeq 267 1401 1721 2000 92.7% chr11 - 114912598 114912922 325 browser details YourSeq 267 1401 1720 2000 93.3% chr14 + 118808124 118808449 326 browser details YourSeq 266 1402 1721 2000 93.5% chr10 - 61797005 61797325 321 browser details YourSeq 266 1401 1720 2000 93.0% chr1 + 136246395 136246720 326 browser details YourSeq 265 1401 1720 2000 93.0% chr16 - 90416878 90417203 326 browser details YourSeq 265 1401 1722 2000 92.1% chr13 - 41691666 41691992 327 browser details YourSeq 265 1400 1718 2000 92.7% chr1 - 35297039 35297362 324

Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rit2 Ras-like without CAAX 2 [ Mus musculus (house mouse) ] Gene ID: 19762, updated on 12-Aug-2019

Gene summary

Official Symbol Rit2 provided by MGI Official Full Name Ras-like without CAAX 2 provided by MGI Primary source MGI:MGI:108054 See related Ensembl:ENSMUSG00000057455 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 Rin; RIBA; Roc2 Expression Biased expression in cerebellum adult (RPKM 36.4), CNS E18 (RPKM 18.8) and 4 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 B1 See Rit2 in Genome Data Viewer Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (30974314..31317128, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (31133968..31476782, complement)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 4 transcripts

Gene: Rit2 ENSMUSG00000057455

Description Ras-like without CAAX 2 [Source:MGI Symbol;Acc:MGI:108054] Gene Synonyms Rin, Roc2 Location Chromosome 18: 30,973,489-31,317,290 reverse strand. GRCm38:CM001011.2 About this gene This gene has 4 transcripts (splice variants), 119 orthologues, 35 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rit2- ENSMUST00000153060.7 1806 217aa ENSMUSP00000114323.1 Protein coding CCDS29108 P70425 TSL:1 203 GENCODE basic APPRIS P1

Rit2- ENSMUST00000139924.1 1674 97aa ENSMUSP00000122938.1 Protein coding - F2Z432 TSL:1 202 GENCODE basic

Rit2- ENSMUST00000082070.12 2715 77aa ENSMUSP00000080724.6 Nonsense mediated - F8WGU6 TSL:1 201 decay

Rit2- ENSMUST00000153196.1 701 No - lncRNA - - TSL:5 204 protein

363.80 kb Forward strand 31.0Mb 31.1Mb 31.2Mb 31.3Mb Contigs AC102497.7 > AC158499.5 > AC102597.22 > AC164011.2 > Genes (Comprehensive set... < Rit2-201nonsense mediated decay

< Rit2-203protein coding

< Rit2-204lncRNA

< Rit2-202protein coding

Regulatory Build

31.0Mb 31.1Mb 31.2Mb 31.3Mb Reverse strand 363.80 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000153060

< Rit2-203protein coding

Reverse strand 342.82 kb

ENSMUSP00000114... Low complexity (Seg) TIGRFAM Small GTP-binding protein domain Superfamily P-loop containing nucleoside triphosphate hydrolase SMART SM00175

SM00173

SM00176

SM00174 Prints PR00449 Pfam Small GTPase PROSITE profiles Small GTPase superfamily, Ras-type PANTHER PTHR24070:SF203

Small GTPase superfamily, Ras-type Gene3D 3.40.50.300 CDD cd04141

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

Variant Legend splice region variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 217

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

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