https://www.alphaknockout.com

Mouse Eno2 Knockout Project (CRISPR/Cas9)

Objective: To create a Eno2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Eno2 gene (NCBI Reference Sequence: NM_013509 ; Ensembl: ENSMUSG00000004267 ) is located on Mouse chromosome 6. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000004378). Exon 2~12 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 2 starts from about 0.08% of the coding region. Exon 2~12 covers 100.0% of the coding region. The size of effective KO region: ~7025 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 5 6 7 8 9 10 11 12

Legends Exon of mouse Eno2 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 start codon 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 stop codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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

Sequence 12

Summary: Full Length(2000bp) | A(17.35% 347) | C(33.25% 665) | T(24.4% 488) | G(25.0% 500)

Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

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

Sequence 12

Summary: Full Length(2000bp) | A(22.9% 458) | C(26.7% 534) | T(26.7% 534) | G(23.7% 474)

Note: The 2000 bp section downstream of stop codon 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% chr6 - 124768323 124770322 2000 browser details YourSeq 160 158 445 2000 86.6% chr4 + 141727234 141727514 281 browser details YourSeq 37 813 865 2000 93.1% chr2 - 163918898 163918959 62 browser details YourSeq 36 394 503 2000 95.0% chr1 - 35756068 35756273 206 browser details YourSeq 31 818 856 2000 89.8% chr17 - 85533640 85533678 39 browser details YourSeq 28 827 854 2000 100.0% chr7 - 12602962 12602989 28 browser details YourSeq 25 861 886 2000 100.0% chr1 - 182112499 182112526 28 browser details YourSeq 22 1301 1322 2000 100.0% chr9 - 122246731 122246752 22 browser details YourSeq 22 831 852 2000 100.0% chr6 - 86211066 86211087 22 browser details YourSeq 22 1951 1972 2000 100.0% chr18 - 16132070 16132091 22 browser details YourSeq 22 1860 1883 2000 87.0% chr10 - 93987804 93987826 23 browser details YourSeq 22 822 843 2000 100.0% chr2 + 35644005 35644026 22 browser details YourSeq 21 1857 1878 2000 100.0% chrY - 342915 342937 23 browser details YourSeq 21 1857 1878 2000 100.0% chr6 - 78752530 78752552 23 browser details YourSeq 21 1301 1322 2000 100.0% chr1 - 24392325 24392347 23 browser details YourSeq 21 1960 1980 2000 100.0% chrX + 73864491 73864511 21 browser details YourSeq 21 1308 1329 2000 100.0% chr5 + 52672936 52672958 23 browser details YourSeq 21 636 656 2000 100.0% chr16 + 91945901 91945921 21 browser details YourSeq 21 827 847 2000 100.0% chr16 + 60249546 60249566 21 browser details YourSeq 20 1303 1322 2000 100.0% chr1 - 41563869 41563888 20

Note: The 2000 bp section upstream of start codon 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% chr6 - 124759296 124761295 2000 browser details YourSeq 110 1107 1242 2000 91.3% chr2 + 174403686 174403819 134 browser details YourSeq 109 1102 1244 2000 91.2% chr1 + 101060735 101060875 141 browser details YourSeq 106 1103 1250 2000 89.6% chr14 + 29989883 29990478 596 browser details YourSeq 106 1117 1251 2000 89.7% chr10 + 117101302 117101434 133 browser details YourSeq 105 1103 1242 2000 88.4% chr15 - 84288833 84288970 138 browser details YourSeq 105 1102 1242 2000 86.3% chr15 + 20906539 20906670 132 browser details YourSeq 105 1102 1242 2000 87.8% chr12 + 84630376 84630514 139 browser details YourSeq 104 1103 1242 2000 90.9% chr7 - 79685130 79685267 138 browser details YourSeq 104 1115 1242 2000 91.6% chr10 + 24732545 24732670 126 browser details YourSeq 103 1048 1227 2000 89.1% chr12 + 112696499 112696673 175 browser details YourSeq 102 1102 1242 2000 85.3% chr6 - 37194021 37194150 130 browser details YourSeq 102 1097 1247 2000 89.9% chr19 + 46252661 46252833 173 browser details YourSeq 101 1102 1242 2000 86.5% chr14 + 122306126 122306257 132 browser details YourSeq 100 1102 1227 2000 91.0% chr15 + 81417665 81417799 135 browser details YourSeq 100 1115 1245 2000 88.6% chr14 + 60303735 60303858 124 browser details YourSeq 99 1102 1242 2000 86.6% chr10 - 81938242 81938375 134 browser details YourSeq 99 1102 1242 2000 86.6% chr10 - 81778901 81779034 134 browser details YourSeq 99 1103 1227 2000 90.5% chr15 + 96793107 96793229 123 browser details YourSeq 99 1104 1242 2000 85.7% chr14 + 34563796 34563934 139

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

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Gene and protein information: Eno2 enolase 2, gamma neuronal [ Mus musculus (house mouse) ] Gene ID: 13807, updated on 24-Oct-2019

Gene summary

Official Symbol Eno2 provided by MGI Official Full Name enolase 2, gamma neuronal provided by MGI Primary source MGI:MGI:95394 See related Ensembl:ENSMUSG00000004267 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 NSE; Eno-2; AI837106; D6Ertd375e Expression Biased expression in cerebellum adult (RPKM 147.0), frontal lobe adult (RPKM 120.3) and 4 other tissuesS ee more Orthologs human all

Genomic context

Location: 6 F2; 6 59.17 cM See Eno2 in Genome Data Viewer Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (124760053..124769673, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (124710073..124719527, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 16 transcripts

Gene: Eno2 ENSMUSG00000004267

Description enolase 2, gamma neuronal [Source:MGI Symbol;Acc:MGI:95394] Gene Synonyms D6Ertd375e, Eno-2, NSE Location Chromosome 6: 124,760,053-124,769,673 reverse strand. GRCm38:CM000999.2 About this gene This gene has 16 transcripts (splice variants), 248 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Eno2- ENSMUST00000004378.14 2795 434aa ENSMUSP00000004378.8 Protein coding CCDS20527 P17183 TSL:1 201 Q545V3 GENCODE basic APPRIS P1

Eno2- ENSMUST00000112476.7 2386 315aa ENSMUSP00000108095.1 Protein coding CCDS80609 D3Z6E4 TSL:1 202 GENCODE basic

Eno2- ENSMUST00000204896.2 1640 391aa ENSMUSP00000144861.1 Protein coding - A0A0N4SUX5 TSL:5 216 GENCODE basic

Eno2- ENSMUST00000149652.1 804 162aa ENSMUSP00000119112.1 Protein coding - D3Z2S4 CDS 3' 213 incomplete TSL:2

Eno2- ENSMUST00000156033.1 622 58aa ENSMUSP00000144698.1 Protein coding - A0A0N4SUI6 CDS 3' 215 incomplete TSL:5

Eno2- ENSMUST00000135626.7 419 121aa ENSMUSP00000114500.1 Protein coding - D3YVD3 CDS 3' 207 incomplete TSL:3

Eno2- ENSMUST00000151214.7 745 37aa ENSMUSP00000145131.1 Nonsense mediated - A0A0N4SUW8 TSL:5 214 decay

Eno2- ENSMUST00000138770.7 616 37aa ENSMUSP00000144854.1 Nonsense mediated - A0A0N4SUW8 TSL:3 208 decay

Eno2- ENSMUST00000127274.7 565 37aa ENSMUSP00000145216.1 Nonsense mediated - A0A0N4SUW8 TSL:3 204 decay

Eno2- ENSMUST00000147307.1 2614 No - Retained intron - - TSL:2 212 protein

Eno2- ENSMUST00000125436.7 2491 No - Retained intron - - TSL:1 203 protein

Eno2- ENSMUST00000139763.7 898 No - Retained intron - - TSL:3 209 protein

Eno2- ENSMUST00000128701.7 728 No - Retained intron - - TSL:3 206 protein

Eno2- ENSMUST00000146712.1 632 No - Retained intron - - TSL:2 210 protein

Eno2- ENSMUST00000147004.1 490 No - Retained intron - - TSL:2 211 protein

Eno2- ENSMUST00000128598.1 716 No - lncRNA - - TSL:3 205 protein

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29.62 kb Forward strand

124.755Mb 124.760Mb 124.765Mb 124.770Mb 124.775Mb Contigs AC164157.3 > Genes (Comprehensive set... < Atn1-201protein coding < Eno2-212retained intron < Eno2-214nonsense mediated decay < Lrrc23-203protein coding

< Atn1-203protein coding < Eno2-201protein coding < Lrrc23-201protein coding

< Gm45234-201nonsense mediated decay < Eno2-202protein coding < Lrrc23-202protein coding

< Eno2-203retained intron < Lrrc23-204retained intron < Lrrc23-205protein coding

< Eno2-216protein coding

< Eno2-209retained intron < Eno2-204nonsense mediated decay

< Eno2-210retained intron < Eno2-213protein coding

< Eno2-205lncRNA < Eno2-206retained intron

< Eno2-208nonsense mediated decay

< Eno2-207protein coding

< Eno2-211retained intron

< Eno2-215protein coding

Regulatory Build

124.755Mb 124.760Mb 124.765Mb 124.770Mb 124.775Mb Reverse strand 29.62 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000004378

< Eno2-201protein coding

Reverse strand 9.62 kb

ENSMUSP00000004... TIGRFAM Enolase Superfamily SSF54826 Enolase-like, C-terminal domain superfamily

SMART Enolase, N-terminal Enolase, C-terminal TIM barrel domain

SFLD SFLDF00002

Enolase Prints Enolase Pfam Enolase, N-terminal Enolase, C-terminal TIM barrel domain

PROSITE patterns Enolase, conserved site PIRSF Enolase PANTHER Enolase

PTHR11902:SF10 HAMAP Enolase Gene3D Enolase-like, N-terminal Enolase-like, C-terminal domain superfamily

CDD Enolase

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 434

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

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