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Mouse Alx3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Alx3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Alx3 gene (NCBI Reference Sequence: NM_007441 ; Ensembl: ENSMUSG00000014603 ) is located on Mouse chromosome 3. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000014747). 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 Alx3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-89L13 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: Homozygous mutation of this gene results in defects in glucose metabolism. Mice homozygous for a reporter allele exhibit partial preweaning lethality, open neural tube and craniofacial defects in some mice.

Exon 2 starts from about 27.02% 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: 5001 bp, and the size of intron 2 for 3'-loxP site insertion: 3500 bp. The size of effective cKO region: ~817 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 2 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Alx3 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. 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(7317bp) | A(25.34% 1854) | C(25.04% 1832) | T(23.0% 1683) | G(26.62% 1948)

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% chr3 + 107597205 107600204 3000 browser details YourSeq 50 1711 1794 3000 92.9% chr6 - 17622165 17623106 942 browser details YourSeq 50 1734 1796 3000 82.8% chr4 - 117423926 117423983 58 browser details YourSeq 50 1736 1796 3000 98.2% chr12 - 72528181 72528243 63 browser details YourSeq 40 1753 1796 3000 88.1% chr13 - 105942736 105942777 42 browser details YourSeq 40 1738 1787 3000 93.9% chr1 + 108747120 108747177 58 browser details YourSeq 32 1758 1796 3000 86.5% chr12 + 4816129 4816166 38 browser details YourSeq 29 1971 2022 3000 94.0% chr11 + 88300786 88300837 52 browser details YourSeq 24 269 294 3000 96.2% chrX + 7586644 7586669 26 browser details YourSeq 22 267 288 3000 100.0% chr9 - 103108261 103108282 22 browser details YourSeq 22 1753 1775 3000 100.0% chr4 - 104912676 104912702 27 browser details YourSeq 22 267 288 3000 100.0% chr9 + 58544316 58544337 22 browser details YourSeq 22 265 288 3000 87.0% chr4 + 88188334 88188356 23 browser details YourSeq 21 267 287 3000 100.0% chr18 - 73809621 73809641 21 browser details YourSeq 21 1194 1214 3000 100.0% chr2 + 27411986 27412006 21 browser details YourSeq 20 269 288 3000 100.0% chr1 + 37606864 37606883 20

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% chr3 + 107601022 107604021 3000 browser details YourSeq 212 2128 2350 3000 96.4% chr2 - 167624301 167624521 221 browser details YourSeq 211 2128 2344 3000 99.1% chr4 - 123359280 123359505 226 browser details YourSeq 209 2132 2352 3000 97.8% chr12 - 71003635 71003901 267 browser details YourSeq 207 2133 2377 3000 97.7% chr6 - 65025236 65025773 538 browser details YourSeq 206 2128 2338 3000 99.1% chr3 + 96735799 96736012 214 browser details YourSeq 205 2134 2342 3000 99.6% chr10 + 76072878 76073095 218 browser details YourSeq 204 2053 2330 3000 93.0% chr5 + 121071849 121072086 238 browser details YourSeq 202 2132 2342 3000 98.6% chr17 - 85439050 85439266 217 browser details YourSeq 202 2128 2332 3000 99.6% chr12 - 108752735 108752947 213 browser details YourSeq 201 2130 2342 3000 98.1% chr14 + 57693328 57693543 216 browser details YourSeq 201 2128 2331 3000 99.6% chr1 + 84971635 84971842 208 browser details YourSeq 200 2128 2332 3000 99.1% chr4 - 139050000 139050220 221 browser details YourSeq 200 2127 2345 3000 98.1% chr17 - 63438141 63438389 249 browser details YourSeq 200 2127 2350 3000 98.1% chr4 + 116025501 116025734 234 browser details YourSeq 200 2128 2332 3000 99.1% chr18 + 75066220 75066440 221 browser details YourSeq 200 2126 2331 3000 99.1% chr10 + 57466400 57466610 211 browser details YourSeq 199 2128 2338 3000 98.1% chr5 - 52713191 52713405 215 browser details YourSeq 199 2128 2331 3000 99.1% chr3 - 37468836 37469043 208 browser details YourSeq 199 2045 2331 3000 97.7% chr1 - 58384148 58384553 406

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

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Gene and protein information: Alx3 aristaless-like homeobox 3 [ Mus musculus (house mouse) ] Gene ID: 11694, updated on 11-Sep-2019

Gene summary

Official Symbol Alx3 provided by MGI Official Full Name aristaless-like homeobox 3 provided by MGI Primary source MGI:MGI:1277097 See related Ensembl:ENSMUSG00000014603 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Summary This gene belongs to Group 1 of aristaless-like genes, which are characterized by the presence of an aristaless domain and Expression a conserved paired-like homeodomain. The encoded protein acts as a transcriptional regulator. The protein plays a role in the development of craniofacial and appendicular skeleton and may have a role in pancreatic function. [provided by RefSeq, Apr 2013] Orthologs Biased expression in limb E14.5 (RPKM 3.6), CNS E11.5 (RPKM 2.2) and 6 other tissues See more human all

Genomic context

Location: 3 F2.3; 3 46.83 cM See Alx3 in Genome Data Viewer Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (107595031..107605875)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (107397949..107408687)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 2 transcripts

Gene: Alx3 ENSMUSG00000014603

Description aristaless-like homeobox 3 [Source:MGI Symbol;Acc:MGI:1277097] Location Chromosome 3: 107,595,031-107,605,776 forward strand. GRCm38:CM000996.2 About this gene This gene has 2 transcripts (splice variants), 165 orthologues, 49 paralogues, is a member of 1 Ensembl protein family and is associated with 21 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Alx3-201 ENSMUST00000014747.2 1874 343aa ENSMUSP00000014747.1 Protein coding CCDS17739 O70137 Q3UQX2 TSL:1 GENCODE basic APPRIS P1

Alx3-202 ENSMUST00000233202.1 564 131aa ENSMUSP00000156514.1 Protein coding - A0A3B2WCG6 CDS 3' incomplete

30.75 kb Forward strand 107.59Mb 107.60Mb 107.61Mb Genes (Comprehensive set... Alx3-201 >protein coding

Alx3-202 >protein coding

Contigs AC122902.4 >

Genes < Strip1-205retained intron (Comprehensive set...

< Strip1-201protein coding

< Strip1-202lncRNA

Regulatory Build

107.59Mb 107.60Mb 107.61Mb Reverse strand 30.75 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000014747

10.75 kb Forward strand

Alx3-201 >protein coding

ENSMUSP00000014... MobiDB lite Low complexity (Seg) Superfamily Homeobox-like domain superfamily SMART Homeobox domain Pfam Homeobox domain PROSITE profiles Homeobox domain PROSITE patterns Homeobox, conserved site PANTHER Homeobox protein aristaless-like 3

PTHR24329 Gene3D 1.10.10.60 CDD Homeobox domain

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 343

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

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