https://www.alphaknockout.com

Mouse Fez1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Fez1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fez1 gene (NCBI Reference Sequence: NM_183171 ; Ensembl: ENSMUSG00000032118 ) is located on Mouse chromosome 9. 9 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000163816). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fez1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-293P19 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 null allele exhibit hyperactivity and increased sensitivity to methamphetamine.

Exon 2 starts from about 26.53% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 6329 bp, and the size of intron 2 for 3'-loxP site insertion: 10365 bp. The size of effective cKO region: ~600 bp. The cKO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fez1 Homology arm cKO region loxP site

Page 2 of 8 https://www.alphaknockout.com

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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7100bp) | A(27.03% 1919) | C(21.32% 1514) | T(29.86% 2120) | G(21.79% 1547)

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.

Page 3 of 8 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr9 + 36847098 36850097 3000 browser details YourSeq 179 240 1036 3000 94.6% chr7 - 79199873 79353748 153876 browser details YourSeq 130 240 1040 3000 90.6% chr10 - 11940741 12016347 75607 browser details YourSeq 107 232 346 3000 94.8% chr5 - 99445834 99445947 114 browser details YourSeq 105 911 1079 3000 85.7% chr13 - 21220602 21220768 167 browser details YourSeq 103 238 344 3000 98.2% chr12 + 106409711 106409817 107 browser details YourSeq 101 240 344 3000 98.1% chr6 - 32639660 32639764 105 browser details YourSeq 101 240 344 3000 98.1% chr12 - 23209170 23209274 105 browser details YourSeq 101 240 344 3000 98.1% chr6 + 124989108 124989212 105 browser details YourSeq 101 240 344 3000 98.1% chr4 + 8900232 8900336 105 browser details YourSeq 101 240 344 3000 98.1% chr3 + 140743712 140743816 105 browser details YourSeq 101 240 344 3000 98.1% chr2 + 21415927 21416031 105 browser details YourSeq 101 240 344 3000 98.1% chr17 + 77033848 77033952 105 browser details YourSeq 101 240 344 3000 98.1% chr15 + 14524268 14524372 105 browser details YourSeq 101 240 344 3000 98.1% chr14 + 22879279 22879383 105 browser details YourSeq 101 240 344 3000 98.1% chr12 + 15184794 15184898 105 browser details YourSeq 101 240 344 3000 98.1% chr11 + 40771729 40771833 105 browser details YourSeq 101 240 344 3000 99.1% chr10 + 123694388 124134787 440400 browser details YourSeq 101 240 344 3000 98.1% chr10 + 83875151 83875255 105 browser details YourSeq 99 240 344 3000 97.2% chr5 - 85550771 85550875 105

Note: The 3000 bp section upstream of Exon 2 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% chr9 + 36850698 36853697 3000 browser details YourSeq 565 2349 2999 3000 96.1% chr6 - 90875861 90876566 706 browser details YourSeq 562 2350 2999 3000 94.3% chr1 - 182704098 182704723 626 browser details YourSeq 557 2383 3000 3000 95.5% chr1 - 143841863 144236237 394375 browser details YourSeq 555 2382 3000 3000 94.7% chrX - 51309562 51310165 604 browser details YourSeq 555 2351 3000 3000 94.1% chr9 - 55653623 55654233 611 browser details YourSeq 555 2382 3000 3000 94.7% chr7 - 18059730 18060333 604 browser details YourSeq 555 2352 3000 3000 94.6% chr4 + 85017204 85017830 627 browser details YourSeq 555 2353 3000 3000 94.6% chr18 + 9536802 9537846 1045 browser details YourSeq 554 2365 3000 3000 94.0% chr16 - 97630443 97631056 614 browser details YourSeq 553 2382 2999 3000 94.7% chr12 - 40878388 40878985 598 browser details YourSeq 553 2383 3000 3000 96.0% chr16 + 76784291 76784919 629 browser details YourSeq 552 2360 3000 3000 94.3% chrX - 95406123 95406753 631 browser details YourSeq 551 2382 3000 3000 94.6% chr8 + 109506435 109507046 612 browser details YourSeq 551 2375 2998 3000 94.2% chr2 + 117111333 117111937 605 browser details YourSeq 550 2382 2999 3000 94.5% chr6 - 106869100 106869695 596 browser details YourSeq 550 2384 3000 3000 95.1% chr4 - 7958439 7959037 599 browser details YourSeq 550 2380 3000 3000 93.9% chr10 - 47790926 47791531 606 browser details YourSeq 550 2381 3000 3000 95.5% chr3 + 10514637 10515254 618 browser details YourSeq 548 2350 3000 3000 93.7% chr8 - 53703856 53704478 623

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 https://www.alphaknockout.com

Gene and protein information: Fez1 fasciculation and elongation protein zeta 1 (zygin I) [ Mus musculus (house mouse) ] Gene ID: 235180, updated on 24-Oct-2019

Gene summary

Official Symbol Fez1 provided by MGI Official Full Name fasciculation and elongation protein zeta 1 (zygin I) provided by MGI Primary source MGI:MGI:2670976 See related Ensembl:ENSMUSG00000032118 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 UNC76; UNC-76 Expression Biased expression in CNS E18 (RPKM 83.7), cerebellum adult (RPKM 71.7) and 6 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 A4 See Fez1 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (36821398..36878926)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (36651244..36686225)

Chromosome 9 - NC_000075.6

Page 5 of 8 https://www.alphaknockout.com

Transcript information: This gene has 10 transcripts

Gene: Fez1 ENSMUSG00000032118

Description fasciculation and elongation protein zeta 1 (zygin I) [Source:MGI Symbol;Acc:MGI:2670976] Gene Synonyms UNC-76, UNC76 Location Chromosome 9: 36,821,864-36,878,924 forward strand. GRCm38:CM001002.2 About this gene This gene has 10 transcripts (splice variants), 205 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fez1- ENSMUST00000034630.14 1794 392aa ENSMUSP00000034630.8 Protein coding CCDS40581 Q8K0X8 TSL:1 201 GENCODE basic APPRIS P1

Fez1- ENSMUST00000163816.7 1285 392aa ENSMUSP00000126072.1 Protein coding CCDS40581 Q8K0X8 TSL:1 208 GENCODE basic APPRIS P1

Fez1- ENSMUST00000161978.1 1519 53aa ENSMUSP00000125475.1 Protein coding - F7BWM9 CDS 5' 205 incomplete TSL:1

Fez1- ENSMUST00000162633.8 927 123aa ENSMUSP00000124634.1 Protein coding - E0CXY1 CDS 3' 207 incomplete TSL:5

Fez1- ENSMUST00000161500.7 748 167aa ENSMUSP00000123762.1 Protein coding - E0CYY9 CDS 3' 204 incomplete TSL:5

Fez1- ENSMUST00000214772.1 419 78aa ENSMUSP00000149910.1 Protein coding - A0A1L1SSG8 CDS 3' 209 incomplete TSL:3

Fez1- ENSMUST00000162235.1 224 47aa ENSMUSP00000124185.1 Protein coding - F6TRI8 CDS 5' 206 incomplete TSL:5

Fez1- ENSMUST00000160041.1 755 91aa ENSMUSP00000124648.1 Nonsense mediated - F6SG45 CDS 5' 203 decay incomplete TSL:3

Fez1- ENSMUST00000159137.1 755 No - Retained intron - - TSL:2 202 protein

Fez1- ENSMUST00000216539.1 303 No - lncRNA - - TSL:5 210 protein

Page 6 of 8 https://www.alphaknockout.com

77.06 kb Forward strand

Genes (Comprehensive set... Fez1-207 >protein coding Fez1-206 >protein coding

Fez1-201 >protein coding

Fez1-209 >protein coding Fez1-202 >retained intron

Fez1-210 >lncRNA Fez1-205 >protein coding

Fez1-204 >protein coding

Fez1-208 >protein coding

Fez1-203 >nonsense mediated decay

Contigs CT955982.10 > CT009699.13 > Genes < Gm27219-201processed pseudogene (Comprehensive set...

Regulatory Build

Reverse strand 77.06 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript pseudogene

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000163816

34.98 kb Forward strand

Fez1-208 >protein coding

ENSMUSP00000126... MobiDB lite Low complexity (Seg) Pfam Fasciculation and elongation protein zeta, FEZ PANTHER Fasciculation and elongation protein zeta 1

Fasciculation and elongation protein zeta, FEZ

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 392

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

Page 8 of 8