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Mouse Plxnb2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Plxnb2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plxnb2 gene (NCBI Reference Sequence: NM_138749 ; Ensembl: ENSMUSG00000036606 ) is located on Mouse chromosome 15. 38 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 38 (Transcript: ENSMUST00000109331). Exon 10~12 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Plxnb2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-63F9 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: Homozygotes for a targeted mutation of this gene die perinatally of exencephaly or survive and seem normal despite severe abnormalities in cerebellar layering and foliation; the external granule cell layer is disorganized due to continued proliferation and migration of differentiated granule cells.

Exon 10 starts from about 32.07% of the coding region. The knockout of Exon 10~12 will result in frameshift of the gene. The size of intron 9 for 5'-loxP site insertion: 712 bp, and the size of intron 12 for 3'-loxP site insertion: 641 bp. The size of effective cKO region: ~1023 bp.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 38 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Plxnb2 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(7523bp) | A(21.32% 1604) | C(28.83% 2169) | T(21.85% 1644) | G(27.99% 2106)

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% chr15 - 89164785 89167784 3000 browser details YourSeq 27 1576 1613 3000 96.6% chr7 - 113082109 113082147 39 browser details YourSeq 24 1712 1735 3000 100.0% chr4 - 130810650 130810673 24 browser details YourSeq 23 1582 1612 3000 87.1% chr1 + 40332170 40332200 31 browser details YourSeq 22 2191 2212 3000 100.0% chr6 - 103900701 103900722 22 browser details YourSeq 22 884 905 3000 100.0% chr9 + 92695976 92695997 22 browser details YourSeq 21 1540 1560 3000 100.0% chr2 - 79591916 79591936 21 browser details YourSeq 21 1245 1265 3000 100.0% chr15 - 96031181 96031201 21 browser details YourSeq 21 884 904 3000 100.0% chr1 - 59862376 59862396 21 browser details YourSeq 21 884 904 3000 100.0% chr7 + 58982016 58982036 21 browser details YourSeq 20 2870 2891 3000 95.5% chr1 + 17842541 17842562 22

Note: The 3000 bp section upstream of Exon 10 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% chr15 - 89160762 89163761 3000 browser details YourSeq 126 38 221 3000 88.9% chr14 - 55417984 55418178 195 browser details YourSeq 117 28 221 3000 87.1% chr2 + 125712404 125712594 191 browser details YourSeq 116 15 188 3000 88.4% chrX - 48582155 48582611 457 browser details YourSeq 111 34 221 3000 87.8% chr7 + 118194644 118194832 189 browser details YourSeq 106 33 225 3000 92.2% chr5 - 88662032 88662232 201 browser details YourSeq 105 34 221 3000 91.4% chr8 - 125290575 125290777 203 browser details YourSeq 105 34 221 3000 91.4% chr19 - 55799404 55799601 198 browser details YourSeq 105 33 221 3000 89.6% chr15 + 93114771 93115011 241 browser details YourSeq 102 34 220 3000 88.1% chr10 - 53372964 53373157 194 browser details YourSeq 102 35 217 3000 84.8% chr4 + 149387706 149387884 179 browser details YourSeq 102 38 221 3000 89.3% chr17 + 27215286 27416489 201204 browser details YourSeq 102 14 221 3000 88.7% chr17 + 27239702 27239923 222 browser details YourSeq 101 40 220 3000 87.4% chrX + 94207714 94207895 182 browser details YourSeq 100 25 220 3000 86.7% chr18 - 80394780 80394990 211 browser details YourSeq 99 34 219 3000 90.2% chr9 - 121852699 121852881 183 browser details YourSeq 96 34 221 3000 88.8% chr18 - 78507845 78508039 195 browser details YourSeq 96 34 221 3000 93.7% chr10 - 80098036 80098245 210 browser details YourSeq 96 40 219 3000 88.2% chr8 + 34070568 34070765 198 browser details YourSeq 96 6 221 3000 89.4% chr2 + 21032784 21033006 223

Note: The 3000 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Plxnb2 plexin B2 [ Mus musculus (house mouse) ] Gene ID: 140570, updated on 12-Aug-2019

Gene summary

Official Symbol Plxnb2 provided by MGI Official Full Name plexin B2 provided by MGI Primary source MGI:MGI:2154239 See related Ensembl:ENSMUSG00000036606 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 Debt; plexin-B2 Expression Ubiquitous expression in colon adult (RPKM 85.9), ovary adult (RPKM 65.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 15 E3; 15 44.68 cM See Plxnb2 in Genome Data Viewer

Exon count: 40

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (89155546..89182018, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (88985979..89011218, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 8 transcripts

Gene: Plxnb2 ENSMUSG00000036606

Description plexin B2 [Source:MGI Symbol;Acc:MGI:2154239] Gene Synonyms 1110007H23Rik, Debt Location Chromosome 15: 89,155,549-89,180,788 reverse strand. GRCm38:CM001008.2 About this gene This gene has 8 transcripts (splice variants), 283 orthologues, 8 paralogues, is a member of 1 Ensembl protein family and is associated with 49 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Plxnb2-202 ENSMUST00000109331.8 6497 1842aa ENSMUSP00000104955.1 Protein coding CCDS49698 B2RXS4 TSL:5 GENCODE basic APPRIS P1

Plxnb2-201 ENSMUST00000060808.9 6394 1842aa ENSMUSP00000051731.9 Protein coding CCDS49698 B2RXS4 TSL:5 GENCODE basic APPRIS P1

Plxnb2-207 ENSMUST00000229966.1 860 No protein - Retained intron - - -

Plxnb2-208 ENSMUST00000230393.1 825 No protein - Retained intron - - -

Plxnb2-206 ENSMUST00000151418.1 732 No protein - Retained intron - - TSL:2

Plxnb2-203 ENSMUST00000131062.1 605 No protein - Retained intron - - TSL:2

Plxnb2-204 ENSMUST00000139372.1 426 No protein - Retained intron - - TSL:2

Plxnb2-205 ENSMUST00000143014.2 834 No protein - lncRNA - - TSL:3

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45.24 kb Forward strand 89.15Mb 89.16Mb 89.17Mb 89.18Mb 89.19Mb Contigs AC113069.10 > < AC160538.8 Genes < Mapk11-204retained intron < Plxnb2-201protein coding < Dennd6b-203nonsense mediated decay (Comprehensive set...

< Mapk11-201protein coding < Plxnb2-202protein coding < Dennd6b-201protein coding

< Mapk11-203lncRNA < Plxnb2-205lncRNA < Plxnb2-204retained intron < Dennd6b-204retained intron

< Mapk11-202retained intron < Plxnb2-207retained intron< Mir7118-201miRNA < Dennd6b-205retained intron

< Plxnb2-206retained intron < Plxnb2-203retained intron < Dennd6b-202retained intron

< Plxnb2-208retained intron < Dennd6b-206retained intron

< Mir6958-201miRNA

Regulatory Build

89.15Mb 89.16Mb 89.17Mb 89.18Mb 89.19Mb Reverse strand 45.24 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000109331

< Plxnb2-202protein coding

Reverse strand 25.24 kb

ENSMUSP00000104... PDB-ENSP mappings Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... Superfamily Sema domain superfamily SSF103575 Immunoglobulin E-set Rho GTPase activation protein

SMART Sema domain PSI domain IPT domain Pfam Sema domain Plexin, TIG domain 1 Plexin, cytoplasmic RasGAP domain

Plexin repeat IPT domain PROSITE profiles Sema domain PANTHER PTHR22625:SF9

Plexin family Gene3D WD40/YVTN repeat-like-containing domain superfamily 3.10.20.90

Immunoglobulin-like fold 1.10.506.10 CDD cd01179 cd12792

cd01180

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

Variant Legend missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1842

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

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