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Mouse Fez2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Fez2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fez2 (NCBI Reference Sequence: NM_001285949 ; Ensembl: ENSMUSG00000056121 ) is located on Mouse 17. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000234052). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fez2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-241H5 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:

Exon 3 starts from about 34.88% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 8019 bp, and the size of intron 4 for 3'-loxP site insertion: 1841 bp. The size of effective cKO region: ~2605 bp. The cKO 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 4 5 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fez2 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(9105bp) | A(24.58% 2238) | C(20.55% 1871) | T(31.72% 2888) | G(23.15% 2108)

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% chr17 - 78405095 78408094 3000 browser details YourSeq 47 696 752 3000 96.1% chr6 - 5193276 5193370 95 browser details YourSeq 47 1208 1305 3000 72.3% chr10 + 72621065 72621146 82 browser details YourSeq 47 690 753 3000 94.4% chr1 + 96570669 96570747 79 browser details YourSeq 43 705 757 3000 93.9% chr13 - 112557435 112557498 64 browser details YourSeq 43 693 746 3000 94.0% chr2 + 118425040 118425104 65 browser details YourSeq 43 689 748 3000 93.7% chr1 + 57306501 57306572 72 browser details YourSeq 42 705 753 3000 97.8% chr13 + 98108896 98108962 67 browser details YourSeq 41 705 753 3000 95.6% chr1 + 188793641 188793771 131 browser details YourSeq 39 705 748 3000 95.5% chr14 + 118807035 118807082 48 browser details YourSeq 38 696 757 3000 76.1% chr2 - 28791301 28791352 52 browser details YourSeq 33 705 746 3000 90.5% chr13 - 89826070 89826115 46 browser details YourSeq 33 714 753 3000 79.5% chr9 + 45248364 45248397 34 browser details YourSeq 32 713 753 3000 90.3% chr10 - 25649003 25649045 43 browser details YourSeq 31 1239 1272 3000 97.1% chr2 - 164455247 164455281 35 browser details YourSeq 31 689 736 3000 89.8% chr3 + 90700350 90700548 199 browser details YourSeq 31 705 747 3000 73.0% chr19 + 54482329 54482365 37 browser details YourSeq 31 708 753 3000 94.2% chr1 + 183867718 183867873 156 browser details YourSeq 29 478 553 3000 96.8% chr2 - 51660316 51660391 76 browser details YourSeq 29 1239 1272 3000 96.8% chr11 - 69787587 69787621 35

Note: The 3000 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 3000 1 3000 3000 100.0% chr17 - 78399490 78402489 3000 browser details YourSeq 30 1384 1420 3000 97.0% chr4 + 122529396 122529746 351 browser details YourSeq 25 1777 1803 3000 88.5% chr13 - 44074596 44074621 26 browser details YourSeq 25 32 57 3000 100.0% chr1 + 74982231 74982257 27 browser details YourSeq 23 32 55 3000 100.0% chr8 + 34668851 34668876 26

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Fez2 fasciculation and elongation protein zeta 2 (zygin II) [ Mus musculus (house mouse) ] Gene ID: 225020, updated on 24-Oct-2019

Gene summary

Official Symbol Fez2 provided by MGI Official Full Name fasciculation and elongation protein zeta 2 (zygin II) provided by MGI Primary source MGI:MGI:2675856 See related Ensembl:ENSMUSG00000056121 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 9030616F16; D17Ertd315e Expression Ubiquitous expression in bladder adult (RPKM 18.4), cerebellum adult (RPKM 9.7) and 28 other tissues See more Orthologs human all

Genomic context

Location: 17 E2; 17 48.57 cM See Fez2 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (78369212..78418152, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (78777225..78817443, complement)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 8 transcripts

Gene: Fez2 ENSMUSG00000056121

Description fasciculation and elongation protein zeta 2 (zygin II) [Source:MGI Symbol;Acc:MGI:2675856] Gene Synonyms D17Ertd315e, zygin 2 Location Chromosome 17: 78,369,212-78,418,152 reverse strand. GRCm38:CM001010.2 About this gene This gene has 8 transcripts (splice variants), 262 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fez2-202 ENSMUST00000112487.2 2041 375aa ENSMUSP00000108106.1 Protein coding CCDS70844 D3Z6D5 TSL:1 GENCODE basic APPRIS ALT1

Fez2-201 ENSMUST00000070039.13 1939 348aa ENSMUSP00000068987.7 Protein coding CCDS28977 Q3U049 Q6TYB5 TSL:1 GENCODE basic APPRIS P3

Fez2-204 ENSMUST00000234052.1 3711 345aa ENSMUSP00000157187.1 Protein coding - Q3TN06 GENCODE basic

Fez2-206 ENSMUST00000234530.1 2001 373aa ENSMUSP00000157338.1 Protein coding - A0A3Q4EH67 GENCODE basic APPRIS ALT1

Fez2-203 ENSMUST00000234029.1 1516 331aa ENSMUSP00000157359.1 Protein coding - A0A3Q4EGS7 GENCODE basic

Fez2-208 ENSMUST00000234900.1 2217 No protein - Retained intron - - -

Fez2-205 ENSMUST00000234067.1 2017 No protein - Retained intron - - -

Fez2-207 ENSMUST00000234601.1 1695 No protein - lncRNA - - -

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68.94 kb Forward strand 78.36Mb 78.38Mb 78.40Mb 78.42Mb Crim1-201 >protein coding Gm50034-201 >lncRNA (Comprehensive set...

Crim1-205 >protein coding

Crim1-203 >protein coding

Contigs < AC103598.6 < AC151270.3

Genes < Fez2-204protein coding (Comprehensive set...

< Fez2-203protein coding

< Fez2-202protein coding

< Fez2-201protein coding

< Fez2-206protein coding

< Fez2-207lncRNA

< Fez2-205retained intron

< Fez2-208retained intron

Regulatory Build

78.36Mb 78.38Mb 78.40Mb 78.42Mb Reverse strand 68.94 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000234052

< Fez2-204protein coding

Reverse strand 48.88 kb

ENSMUSP00000157... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Fasciculation and elongation protein zeta, FEZ

PANTHER Fasciculation and elongation protein zeta, FEZ

Fasciculation and elongation protein zeta 2

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

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 345

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

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