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Mouse Eml1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Eml1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Eml1 gene (NCBI Reference Sequence: NM_001043335 ; Ensembl: ENSMUSG00000058070 ) is located on Mouse chromosome 12. 22 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 22 (Transcript: ENSMUST00000109860). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Eml1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-105K22 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 a spontaneous mutation exhibit subcortical band heterotopia associated with seizures, developmental delay and behavioral deficits.

Exon 6 starts from about 22.32% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 4060 bp, and the size of intron 6 for 3'-loxP site insertion: 2477 bp. The size of effective cKO region: ~630 bp. The cKO 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 6 22 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Eml1 Homology arm 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(7130bp) | A(26.34% 1878) | C(22.99% 1639) | T(27.18% 1938) | G(23.49% 1675)

Note: The sequence of homologous arms and cKO region 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 3000 1 3000 3000 100.0% chr12 + 108503230 108506229 3000 browser details YourSeq 123 1654 2257 3000 77.5% chr11 + 101586690 101587009 320 browser details YourSeq 122 1879 2036 3000 91.9% chr17 + 58425125 58425523 399 browser details YourSeq 120 2115 2262 3000 92.3% chr5 - 103748945 103749092 148 browser details YourSeq 116 1845 2034 3000 84.2% chr2 + 133573793 133573959 167 browser details YourSeq 113 1618 2034 3000 92.1% chr16 - 95730566 95731026 461 browser details YourSeq 111 1845 2021 3000 83.5% chr4 - 142869633 142869791 159 browser details YourSeq 106 2124 2262 3000 90.2% chr17 - 80516876 80517017 142 browser details YourSeq 105 1618 2203 3000 74.4% chr1 + 133095751 133096043 293 browser details YourSeq 102 2125 2243 3000 93.3% chr9 + 95677681 95677800 120 browser details YourSeq 101 1845 2014 3000 83.5% chr11 + 35733250 35733393 144 browser details YourSeq 100 2125 2243 3000 93.2% chr6 + 46361607 46361725 119 browser details YourSeq 98 1854 2020 3000 83.5% chr5 - 91203554 91203693 140 browser details YourSeq 98 1880 2011 3000 94.8% chr1 + 52580960 52581114 155 browser details YourSeq 97 2124 2243 3000 92.2% chr11 - 100574143 100574263 121 browser details YourSeq 97 1874 2034 3000 84.1% chr13 + 95990276 95990419 144 browser details YourSeq 96 1877 2028 3000 92.2% chr3 + 152850831 152851110 280 browser details YourSeq 96 1880 2034 3000 89.1% chr3 + 53274276 53274440 165 browser details YourSeq 93 1621 2204 3000 78.9% chr13 + 54599513 54600059 547 browser details YourSeq 89 1883 2003 3000 92.7% chr15 - 86783321 86783471 151

Note: The 3000 bp section upstream of Exon 6 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% chr12 + 108506860 108509859 3000 browser details YourSeq 428 55 583 3000 92.6% chr10 + 121000877 121001510 634 browser details YourSeq 401 55 596 3000 92.8% chr13 - 106819039 106819805 767 browser details YourSeq 332 55 608 3000 93.8% chr11 - 94960032 94962001 1970 browser details YourSeq 325 57 538 3000 90.5% chr4 + 43235639 43236230 592 browser details YourSeq 324 54 556 3000 91.2% chr8 - 123423381 123424226 846 browser details YourSeq 324 54 608 3000 88.3% chr15 + 85225261 85225659 399 browser details YourSeq 321 54 610 3000 87.6% chr3 + 138707816 138708217 402 browser details YourSeq 316 53 609 3000 87.0% chr9 - 106254726 106255126 401 browser details YourSeq 314 54 443 3000 92.3% chr18 - 62694221 62694617 397 browser details YourSeq 314 55 609 3000 92.3% chr5 + 102380430 102528826 148397 browser details YourSeq 314 55 445 3000 91.8% chr13 + 48423921 48424317 397 browser details YourSeq 312 56 447 3000 91.8% chr5 + 150371764 150372587 824 browser details YourSeq 311 11 608 3000 86.1% chr14 + 78951730 78952155 426 browser details YourSeq 310 57 445 3000 90.7% chr5 - 146326923 146327310 388 browser details YourSeq 310 57 609 3000 87.9% chr3 - 84184454 84184852 399 browser details YourSeq 310 55 647 3000 91.7% chr13 + 46472025 46472635 611 browser details YourSeq 309 55 608 3000 92.1% chr19 + 44353287 44425365 72079 browser details YourSeq 308 55 610 3000 87.4% chr19 - 23670492 23670891 400 browser details YourSeq 308 55 445 3000 92.4% chr16 - 85052027 85052421 395

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: Eml1 echinoderm microtubule associated protein like 1 [ Mus musculus (house mouse) ] Gene ID: 68519, updated on 24-Oct-2019

Gene summary

Official Symbol Eml1 provided by MGI Official Full Name echinoderm microtubule associated protein like 1 provided by MGI Primary source MGI:MGI:1915769 See related Ensembl:ENSMUSG00000058070 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 EMAP; heco; ELP79; EMAPL; EMAP-1; AA171013; AI847476; AI853955; 1110008N23Rik; A930030P13Rik Expression Broad expression in bladder adult (RPKM 32.9), subcutaneous fat pad adult (RPKM 15.9) and 22 other tissues See more Orthologs human all

Genomic context

Location: 12 F1; 12 59.46 cM See Eml1 in Genome Data Viewer

Exon count: 30

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (108371002..108539576)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (109648865..109777774)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 8 transcripts

Gene: Eml1 ENSMUSG00000058070

Description echinoderm microtubule associated protein like 1 [Source:MGI Symbol;Acc:MGI:1915769] Gene Synonyms 1110008N23Rik, A930030P13Rik, ELP79, heco Location Chromosome 12: 108,370,957-108,539,617 forward strand. GRCm38:CM001005.2 About this gene This gene has 8 transcripts (splice variants), 211 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 41 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Eml1- ENSMUST00000109860.7 4160 814aa ENSMUSP00000105486.1 Protein coding CCDS36555 Q05BC3 TSL:1 203 GENCODE basic APPRIS P4

Eml1- ENSMUST00000054955.13 3879 783aa ENSMUSP00000057209.7 Protein coding CCDS36556 Q05BC3 TSL:1 201 GENCODE basic APPRIS ALT2

Eml1- ENSMUST00000109857.7 2627 800aa ENSMUSP00000105483.1 Protein coding CCDS70420 D3Z4J9 TSL:1 202 GENCODE basic APPRIS ALT2

Eml1- ENSMUST00000130999.1 2493 699aa ENSMUSP00000118325.1 Nonsense mediated - D6RII3 TSL:2 205 decay

Eml1- ENSMUST00000138456.7 2984 No - Retained intron - - TSL:1 206 protein

Eml1- ENSMUST00000155544.7 4169 No - lncRNA - - TSL:5 208 protein

Eml1- ENSMUST00000123035.1 1730 No - lncRNA - - TSL:1 204 protein

Eml1- ENSMUST00000148186.1 332 No - lncRNA - - TSL:3 207 protein

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188.66 kb Forward strand 108.40Mb 108.45Mb 108.50Mb Genes (Comprehensive set... Cyp46a1-201 >protein coding Eml1-201 >protein coding

Eml1-206 >retained intron Eml1-207 >lncRNA

Eml1-202 >protein coding

Eml1-203 >protein coding

Eml1-205 >nonsense mediated decay

Eml1-208 >lncRNA

Eml1-204 >lncRNA

Contigs < AC154910.3

Genes < Gm15636-201processed pseudogene < Gm16596-203lncRNA (Comprehensive set...

< Gm16596-201lncRNA

< Gm16596-202lncRNA

Regulatory Build

108.40Mb 108.45Mb 108.50Mb Reverse strand 188.66 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript pseudogene

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Transcript: ENSMUST00000109860

116.75 kb Forward strand

Eml1-203 >protein coding

ENSMUSP00000105... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Quinoprotein alcohol dehydrogenase-like superfamily

SSF50960 SMART WD40 repeat Pfam HELP WD40 repeat

PROSITE profiles WD40-repeat-containing domain

WD40 repeat PROSITE patterns WD40 repeat, conserved site

PANTHER PTHR13720:SF22

PTHR13720 Gene3D WD40/YVTN repeat-like-containing domain superfamily

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

Variant Legend stop lost missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 814

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

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