https://www.alphaknockout.com

Mouse Ythdf3 Knockout Project (CRISPR/Cas9)

Objective: To create a Ythdf3 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ythdf3 (NCBI Reference Sequence: NM_172677 ; Ensembl: ENSMUSG00000047213 ) is located on Mouse 3. 6 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000108346). 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 2.8% of the coding region. Exon 3 covers 4.81% of the coding region. The size of effective KO region: ~86 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 6

Legends Exon of mouse Ythdf3 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(28.95% 579) | C(15.85% 317) | T(36.9% 738) | G(18.3% 366)

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(32.0% 640) | C(12.2% 244) | T(37.2% 744) | G(18.6% 372)

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% chr3 + 16187479 16189478 2000 browser details YourSeq 24 1538 1565 2000 92.9% chr2 + 28874005 28874032 28

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% chr3 + 16189565 16191564 2000 browser details YourSeq 44 1484 1643 2000 90.8% chr12 - 70074423 70074582 160 browser details YourSeq 44 1489 1645 2000 90.8% chr18 + 60765739 60765921 183 browser details YourSeq 43 1474 1646 2000 90.4% chr11 + 97538307 97538492 186 browser details YourSeq 38 1577 1646 2000 80.7% chr9 + 110000911 110000981 71 browser details YourSeq 36 1595 1650 2000 87.8% chrX - 77006280 77006337 58 browser details YourSeq 35 1598 1645 2000 92.7% chr13 + 9364423 9364471 49 browser details YourSeq 32 1615 1652 2000 86.5% chr17 + 48053936 48053972 37 browser details YourSeq 28 851 891 2000 96.7% chr3 + 7669146 7669186 41 browser details YourSeq 28 1615 1648 2000 91.2% chr14 + 101850605 101850638 34 browser details YourSeq 27 1615 1645 2000 93.6% chr7 - 117678861 117678891 31 browser details YourSeq 27 1615 1643 2000 96.6% chr14 - 20800081 20800109 29 browser details YourSeq 27 1615 1645 2000 93.6% chr12 - 86670861 86670891 31 browser details YourSeq 27 1616 1648 2000 91.0% chr18 + 60798324 60798356 33 browser details YourSeq 27 1598 1631 2000 96.6% chr18 + 34082325 34082359 35 browser details YourSeq 27 1615 1645 2000 93.6% chr10 + 29336071 29336101 31 browser details YourSeq 27 1500 1536 2000 96.6% chr10 + 28079433 28079470 38 browser details YourSeq 26 1615 1646 2000 90.7% chr19 - 31157581 31157612 32 browser details YourSeq 26 1615 1646 2000 90.7% chr11 - 117403615 117403646 32 browser details YourSeq 26 1615 1646 2000 90.7% chr16 + 29852840 29852871 32

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 information: Ythdf3 YTH N6-methyladenosine RNA binding protein 3 [ Mus musculus (house mouse) ] Gene ID: 229096, updated on 27-Aug-2019

Gene summary

Official Symbol Ythdf3 provided by MGI Official Full Name YTH N6-methyladenosine RNA binding protein 3 provided by MGI Primary source MGI:MGI:1918850 See related Ensembl:ENSMUSG00000047213 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 9130022A11Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 17.9), limb E14.5 (RPKM 16.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 A1 See Ythdf3 in Genome Data Viewer Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (16183148..16217037)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (16083183..16117037)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 4 transcripts

Gene: Ythdf3 ENSMUSG00000047213

Description YTH N6-methyladenosine RNA binding protein 3 [Source:MGI Symbol;Acc:MGI:1918850] Gene Synonyms 9130022A11Rik Location Chromosome 3: 16,183,212-16,217,037 forward strand. GRCm38:CM000996.2 About this gene This gene has 4 transcripts (splice variants), 199 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ythdf3-201 ENSMUST00000108345.8 5102 585aa ENSMUSP00000103982.2 Protein coding CCDS50869 Q8BYK6 TSL:1 GENCODE basic APPRIS P1

Ythdf3-202 ENSMUST00000108346.4 4929 596aa ENSMUSP00000103983.2 Protein coding CCDS38396 Q8BYK6 TSL:1 GENCODE basic

Ythdf3-203 ENSMUST00000191774.5 3196 589aa ENSMUSP00000141610.1 Protein coding - Q8BYK6 TSL:1 GENCODE basic

Ythdf3-204 ENSMUST00000193598.4 723 No protein - lncRNA - - TSL:5

53.83 kb Forward strand 16.18Mb 16.19Mb 16.20Mb 16.21Mb 16.22Mb (Comprehensive set... Ythdf3-201 >protein coding

Ythdf3-202 >protein coding

Ythdf3-203 >protein coding

Ythdf3-204 >lncRNA

Contigs AC122053.3 > Regulatory Build

16.18Mb 16.19Mb 16.20Mb 16.21Mb 16.22Mb Reverse strand 53.83 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

RNA gene

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Transcript: ENSMUST00000108346

33.62 kb Forward strand

Ythdf3-202 >protein coding

ENSMUSP00000103... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF81995 Pfam YTH domain PROSITE profiles YTH domain PANTHER PTHR12357

PTHR12357:SF9 Gene3D 3.10.590.10

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 596

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

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