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Mouse Etv2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Etv2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Etv2 gene (NCBI Reference Sequence: NM_007959 ; Ensembl: ENSMUSG00000006311 ) is located on Mouse chromosome 7. 6 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000108147). Exon 1~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Etv2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-129B10 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 null alleles die during organogenesis and lack blood vessels. In addition, mice homozygousfor one allele lack endocardial cells, while mice homozygous for another allele lack blood cells.

Exon 1~6 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 6. The size of effective cKO region: ~2725 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele A T

5' G gRNA region 3'

1 2 3 4 5 6

Targeting vector A T G

Targeted allele A T G

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Etv2 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. 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8194bp) | A(26.65% 2184) | C(24.21% 1984) | T(24.59% 2015) | G(24.54% 2011)

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% chr7 - 30635831 30638830 3000 browser details YourSeq 292 226 1500 3000 92.0% chr10 - 59786158 60178366 392209 browser details YourSeq 105 1356 1498 3000 91.0% chr14 + 121690647 121690795 149 browser details YourSeq 100 1335 1501 3000 88.1% chr10 - 119392813 119392981 169 browser details YourSeq 94 226 769 3000 78.0% chr10 + 78378622 78378857 236 browser details YourSeq 87 1335 1498 3000 88.5% chr5 - 115294481 115294634 154 browser details YourSeq 84 1390 1498 3000 89.0% chr1 + 135286853 135286968 116 browser details YourSeq 81 1392 1498 3000 91.8% chr1 - 32123470 32123583 114 browser details YourSeq 81 1388 1498 3000 87.9% chr10 + 30168661 30168779 119 browser details YourSeq 77 207 769 3000 86.0% chr19 + 45133051 45133614 564 browser details YourSeq 76 1367 1497 3000 96.4% chr1 + 106286508 106286645 138 browser details YourSeq 74 206 358 3000 79.1% chr1 - 84617290 84617436 147 browser details YourSeq 74 226 372 3000 87.3% chrX + 160499369 160499514 146 browser details YourSeq 73 210 349 3000 81.0% chr2 - 31019484 31019618 135 browser details YourSeq 72 490 825 3000 71.6% chr18 - 42104605 42104818 214 browser details YourSeq 72 207 349 3000 75.6% chr18 + 24622923 24623066 144 browser details YourSeq 72 226 372 3000 92.9% chr1 + 60137753 60514601 376849 browser details YourSeq 71 1409 1498 3000 90.0% chr11 + 67950636 67950732 97 browser details YourSeq 70 738 861 3000 88.2% chr1 - 13493221 13493537 317 browser details YourSeq 69 1391 1494 3000 89.7% chr1 - 177606555 177606660 106

Note: The 3000 bp section upstream of Exon 1 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% chr7 - 30630637 30633636 3000 browser details YourSeq 347 2559 3000 3000 92.1% chr17 + 83702482 83702932 451 browser details YourSeq 302 2561 3000 3000 91.0% chr17 - 3117345 3117759 415 browser details YourSeq 288 2324 3000 3000 89.6% chr4 - 83320625 83321267 643 browser details YourSeq 287 2270 3000 3000 88.2% chr15 + 33535976 33536443 468 browser details YourSeq 263 2323 3000 3000 93.1% chr1 + 37982842 37983582 741 browser details YourSeq 248 2270 3000 3000 88.3% chr16 - 31339779 31340276 498 browser details YourSeq 247 2559 3000 3000 95.3% chr18 + 67179648 67180090 443 browser details YourSeq 225 2760 3000 3000 97.1% chr19 + 40909668 40909913 246 browser details YourSeq 224 2760 3000 3000 96.7% chr17 + 24368300 24368544 245 browser details YourSeq 222 2760 3000 3000 96.3% chr5 + 147976941 147977185 245 browser details YourSeq 222 2760 3000 3000 96.3% chr17 + 30544531 30544773 243 browser details YourSeq 221 2761 3000 3000 96.3% chr11 - 19885994 19886237 244 browser details YourSeq 221 2558 3000 3000 89.2% chr13 + 91689002 91689247 246 browser details YourSeq 220 2760 3000 3000 95.9% chr7 - 121178330 121178574 245 browser details YourSeq 220 2760 3000 3000 95.9% chr13 + 90800704 90800948 245 browser details YourSeq 220 2726 3000 3000 94.8% chr1 + 54797545 54798272 728 browser details YourSeq 219 2559 3000 3000 88.4% chr8 + 104296085 104296330 246 browser details YourSeq 219 2558 3000 3000 88.1% chr2 + 174520301 174520547 247 browser details YourSeq 219 2556 3000 3000 88.2% chr2 + 72167049 72167297 249

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Etv2 ets variant 2 [ Mus musculus (house mouse) ] Gene ID: 14008, updated on 10-Sep-2019

Gene summary

Official Symbol Etv2 provided by MGI Official Full Name ets variant 2 provided by MGI Primary source MGI:MGI:99253 See related Ensembl:ENSMUSG00000006311 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 Etsrp71 Expression Restricted expression toward testis adult (RPKM 3.1) See more Orthologs human all

Genomic context

Location: 7; 7 B1 See Etv2 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (30633616..30636509, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (31418635..31420871, complement)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 1 transcript

Gene: Etv2 ENSMUSG00000006311

Description ets variant 2 [Source:MGI Symbol;Acc:MGI:99253] Gene Synonyms Etsrp71 Location Chromosome 7: 30,633,616-30,635,852 reverse strand. GRCm38:CM001000.2 About this gene This gene has 1 transcript (splice variant), 84 orthologues, 27 paralogues, is a member of 1 Ensembl protein family and is associated with 18 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Etv2-201 ENSMUST00000108147.2 1048 335aa ENSMUSP00000103782.2 Protein coding CCDS39888 P41163 TSL:1 GENCODE basic APPRIS P1

22.24 kb Forward strand 30.625Mb 30.630Mb 30.635Mb 30.640Mb 30.645Mb Contigs < AC167978.4 Genes (Comprehensive set... < Cox6b1-201protein coding < Etv2-201protein coding < Rbm42-202protein coding

< Cox6b1-204protein coding < Rbm42-201protein coding

< Cox6b1-202retained intron < Gm21982-201retained intron

< Rbm42-203retained intron

Regulatory Build

30.625Mb 30.630Mb 30.635Mb 30.640Mb 30.645Mb Reverse strand 22.24 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000108147

< Etv2-201protein coding

Reverse strand 2.24 kb

ENSMUSP00000103... Low complexity (Seg) Superfamily Winged helix DNA-binding domain superfamily SMART Ets domain Prints Ets domain Pfam Ets domain PROSITE profiles Ets domain

PROSITE patterns Ets domain Ets domain

PANTHER PTHR11849:SF206

PTHR11849 Gene3D Winged helix-like DNA-binding domain superfamily

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 335

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

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