https://www.alphaknockout.com

Mouse Plxna2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Plxna2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plxna2 gene (NCBI Reference Sequence: NM_008882 ; Ensembl: ENSMUSG00000026640 ) is located on Mouse chromosome 1. 32 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 32 (Transcript: ENSMUST00000027952). 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 Plxna2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-81N17 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 knock-out allele show abnormal granule cell migration in the adult cerebellum and aberrant projection of mossy fibers in hippocampal slices. Mice homozygous for an ENU-induced allele are smaller and show granule cell migration defects and mild ataxia with incomplete penetrance.

Exon 2 starts from about 100% 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: 23288 bp, and the size of intron 2 for 3'-loxP site insertion: 4508 bp. The size of effective cKO region: ~1688 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 32 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Plxna2 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(8188bp) | A(28.66% 2347) | C(22.78% 1865) | T(26.36% 2158) | G(22.2% 1818)

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% chr1 + 194640511 194643510 3000

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% chr1 + 194645199 194648198 3000 browser details YourSeq 132 1034 1232 3000 83.5% chr9 + 94648145 94648344 200 browser details YourSeq 130 1037 1227 3000 85.5% chr1 - 138463508 138463724 217 browser details YourSeq 125 1034 1239 3000 85.8% chr17 + 4436165 4436395 231 browser details YourSeq 120 1029 1231 3000 88.1% chr7 + 115141190 115141421 232 browser details YourSeq 119 1034 1212 3000 86.0% chr3 + 32547653 32547834 182 browser details YourSeq 117 1029 1239 3000 87.2% chr4 - 124554466 124554683 218 browser details YourSeq 116 1029 1187 3000 87.2% chr5 - 30726916 30727076 161 browser details YourSeq 116 1023 1208 3000 85.8% chr16 - 97760822 97761008 187 browser details YourSeq 115 1029 1208 3000 89.2% chr4 + 49695917 49696117 201 browser details YourSeq 115 1036 1205 3000 85.3% chr18 + 10373856 10374026 171 browser details YourSeq 113 1034 1225 3000 81.3% chr2 - 180107362 180107556 195 browser details YourSeq 111 1030 1191 3000 87.4% chr11 - 78507975 78508134 160 browser details YourSeq 111 1037 1226 3000 88.8% chr14 + 105471865 105472073 209 browser details YourSeq 110 1034 1205 3000 89.3% chr17 - 12403722 12403922 201 browser details YourSeq 109 1029 1168 3000 89.3% chr15 + 63496248 63496388 141 browser details YourSeq 108 1034 1177 3000 87.5% chr1 - 154568795 154568938 144 browser details YourSeq 108 1035 1168 3000 91.6% chr8 + 34500599 34500732 134 browser details YourSeq 107 1034 1187 3000 85.1% chr4 + 127252840 127252994 155 browser details YourSeq 106 1029 1165 3000 89.6% chrX + 36265986 36266123 138

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: Plxna2 plexin A2 [ Mus musculus (house mouse) ] Gene ID: 18845, updated on 12-Aug-2019

Gene summary

Official Symbol Plxna2 provided by MGI Official Full Name plexin A2 provided by MGI Primary source MGI:MGI:107684 See related Ensembl:ENSMUSG00000026640 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 OCT; Plxn2; PlexA2; AA589422; AW457381; mKIAA0463; 2810428A13Rik Expression Broad expression in CNS E14 (RPKM 20.1), whole brain E14.5 (RPKM 19.4) and 25 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 H6 See Plxna2 in Genome Data Viewer

Exon count: 32

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (194619791..194816869)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (196446023..196643063)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 5 transcripts

Gene: Plxna2 ENSMUSG00000026640

Description plexin A2 [Source:MGI Symbol;Acc:MGI:107684] Gene Synonyms 2810428A13Rik, OCT, PlexA2, Plxn2 Location Chromosome 1: 194,618,218-194,816,869 forward strand. GRCm38:CM000994.2 About this gene This gene has 5 transcripts (splice variants), 266 orthologues, 8 paralogues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Plxna2- ENSMUST00000027952.11 11040 1894aa ENSMUSP00000027952.5 Protein coding CCDS35827 P70207 TSL:1 201 GENCODE basic APPRIS P1

Plxna2- ENSMUST00000135664.1 605 83aa ENSMUSP00000118087.1 Nonsense mediated - F6VSI0 CDS 5' 204 decay incomplete TSL:5

Plxna2- ENSMUST00000124785.1 3180 No - Retained intron - - TSL:1 202 protein

Plxna2- ENSMUST00000194398.1 2650 No - Retained intron - - TSL:NA 205 protein

Plxna2- ENSMUST00000125381.1 3951 No - lncRNA - - TSL:2 203 protein

218.65 kb Forward strand

194.65Mb 194.70Mb 194.75Mb 194.80Mb Genes (Comprehensive set... Plxna2-205 >retained intronGm37783-201 >TEC Gm38036-201 >TEC 2900035J10Rik-201 >TEC Plxna2-202 >retained intron

Plxna2-201 >protein coding

Plxna2-203 >lncRNAGm37674-201 >TEC Plxna2-204 >nonsense mediated decay

Mir3962-201 >miRNA

Contigs AC105073.16 > < AC162447.5 Genes < Gm19777-201processed pseudogene (Comprehensive set...

Regulatory Build

194.65Mb 194.70Mb 194.75Mb 194.80Mb Reverse strand 218.65 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene processed transcript RNA gene

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Transcript: ENSMUST00000027952

197.04 kb Forward strand

Plxna2-201 >protein coding

ENSMUSP00000027... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Sema domain superfamily SSF103575 Immunoglobulin E-set Rho GTPase activation protein

SMART Sema domain PSI domain IPT domain

Pfam Sema domain Plexin repeat IPT domain Plexin, cytoplasmic RasGAP domain

Plexin, TIG domain 1

Plexin, TIG domain 2 PROSITE profiles Sema domain

PANTHER Plexin family

PTHR22625:SF37 Gene3D Immunoglobulin-like fold

WD40/YVTN repeat-like-containing domain superfamily CDD cd11272 cd01180 cd01181 cd12790

cd01179 cd00603

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1894

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

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