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Mouse Unc13b Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Unc13b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Unc13b gene (NCBI Reference Sequence: NM_021468 ; Ensembl: ENSMUSG00000028456 ) is located on Mouse chromosome 4. 40 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 40 (Transcript: ENSMUST00000107952). Exon 14~15 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Unc13b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-309K21 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 mutant mice are grossly phenotypically normal. Mice older than 12 months will exhibit sporadic seizures.

Exon 14 starts from about 27.67% of the coding region. The knockout of Exon 14~15 will result in frameshift of the gene. The size of intron 13 for 5'-loxP site insertion: 2057 bp, and the size of intron 15 for 3'-loxP site insertion: 1518 bp. The size of effective cKO region: ~1236 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 13 14 15 16 40 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Unc13b 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(7736bp) | A(25.32% 1959) | C(22.36% 1730) | T(29.3% 2267) | G(23.01% 1780)

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% chr4 + 43231417 43234416 3000 browser details YourSeq 27 93 133 3000 87.1% chr15 - 12787686 12787725 40 browser details YourSeq 24 86 115 3000 90.0% chr2 - 154773848 154773877 30 browser details YourSeq 24 294 319 3000 88.0% chr13 - 92858538 92858562 25 browser details YourSeq 24 1388 1419 3000 88.5% chr10 + 13340701 13340731 31

Note: The 3000 bp section upstream of Exon 14 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% chr4 + 43235653 43238652 3000 browser details YourSeq 362 2 659 3000 92.0% chr17 + 13788879 13789535 657 browser details YourSeq 341 1 717 3000 89.0% chr5 - 21770493 21771142 650 browser details YourSeq 337 1 666 3000 88.7% chr6 + 85852682 85853142 461 browser details YourSeq 335 1 658 3000 88.9% chr3 - 87578241 87578744 504 browser details YourSeq 330 1 578 3000 92.5% chr5 + 151700666 151702816 2151 browser details YourSeq 329 1 578 3000 88.7% chr16 - 95393683 95394217 535 browser details YourSeq 325 1 386 3000 91.8% chr19 - 20047714 20048096 383 browser details YourSeq 325 1 391 3000 91.4% chr2 + 3497482 3497869 388 browser details YourSeq 325 1 383 3000 92.0% chr10 + 83265104 83265482 379 browser details YourSeq 324 1 384 3000 91.5% chr14 + 72891487 72891866 380 browser details YourSeq 324 3 383 3000 93.4% chr10 + 117321558 117321938 381 browser details YourSeq 324 1 384 3000 91.3% chr1 + 189905025 189905404 380 browser details YourSeq 323 1 385 3000 91.5% chr2 + 19167406 19167787 382 browser details YourSeq 322 1 383 3000 91.8% chr2 + 175648212 175648591 380 browser details YourSeq 322 1 383 3000 91.8% chr15 + 35178761 35179140 380 browser details YourSeq 321 1 383 3000 91.7% chr2 - 175427569 175427948 380 browser details YourSeq 321 1 383 3000 91.2% chr19 - 57332544 57332922 379 browser details YourSeq 321 1 385 3000 92.4% chr14 - 53009221 53009605 385 browser details YourSeq 321 1 384 3000 91.5% chr7 + 49619617 49619997 381

Note: The 3000 bp section downstream of Exon 15 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Unc13b unc-13 homolog B [ Mus musculus (house mouse) ] Gene ID: 22249, updated on 12-Aug-2019

Gene summary

Official Symbol Unc13b provided by MGI Official Full Name unc-13 homolog B provided by MGI Primary source MGI:MGI:1342278 See related Ensembl:ENSMUSG00000028456 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 Unc13a; Unc13h1; Unc13h2; Munc13-1; Munc13-2 Expression Broad expression in CNS E18 (RPKM 6.9), CNS E14 (RPKM 6.8) and 23 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 A5 See Unc13b in Genome Data Viewer

Exon count: 47

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (43046193..43264873)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (43071856..43277759)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 16 transcripts

Gene: Unc13b ENSMUSG00000028456

Description unc-13 homolog B [Source:MGI Symbol;Acc:MGI:1342278] Gene Synonyms Munc13-2, Unc13h2 Location Chromosome 4: 43,058,953-43,264,871 forward strand. GRCm38:CM000997.2 About this gene This gene has 16 transcripts (splice variants), 200 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Unc13b- ENSMUST00000079978.12 6354 1590aa ENSMUSP00000078894.6 Protein CCDS80089 Q9Z1N9 TSL:5 201 coding GENCODE basic APPRIS P2

Unc13b- ENSMUST00000107952.8 5037 1602aa ENSMUSP00000103586.2 Protein CCDS51161 Q9Z1N9 TSL:1 202 coding

Unc13b- ENSMUST00000163653.7 5034 1601aa ENSMUSP00000128608.1 Protein CCDS38738 Q9Z1N9 TSL:1 212 coding

Unc13b- ENSMUST00000207569.1 14754 4390aa ENSMUSP00000147100.1 Protein - A0A140LJ69 TSL:5 215 coding GENCODE basic

Unc13b- ENSMUST00000207708.1 6574 1982aa ENSMUSP00000146589.1 Protein - A0A140LHX5 TSL:5 216 coding GENCODE basic

Unc13b- ENSMUST00000107953.8 5034 1609aa ENSMUSP00000103587.2 Protein - E9Q263 TSL:5 203 coding GENCODE basic APPRIS ALT1

Unc13b- ENSMUST00000168032.1 768 256aa ENSMUSP00000132622.1 Protein - F6X605 CDS 5' and 3' 213 coding incomplete TSL:3

Unc13b- ENSMUST00000145899.1 569 189aa ENSMUSP00000128638.1 Protein - F7CEK4 CDS 5' and 3' 208 coding incomplete TSL:5

Unc13b- ENSMUST00000171234.1 3617 No - Retained - - TSL:1 214 protein intron

Unc13b- ENSMUST00000149945.2 3258 No - Retained - - TSL:2 209 protein intron

Unc13b- ENSMUST00000143653.2 1036 No - Retained - - TSL:5 207 protein intron

Unc13b- ENSMUST00000153168.2 856 No - Retained - - TSL:3 211 protein intron

Unc13b- ENSMUST00000126878.1 712 No - Retained - - TSL:3 204 protein intron

Unc13b- ENSMUST00000127597.1 495 No - Retained - - TSL:3 205 protein intron

Unc13b- ENSMUST00000151611.8 2173 No - lncRNA - - TSL:1 210 protein

Unc13b- ENSMUST00000132310.8 780 No - lncRNA - - TSL:5 206 protein

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225.92 kb Forward strand

43.05Mb 43.10Mb 43.15Mb 43.20Mb 43.25Mb Genes (Comprehensive set... Gm26881-202 >lncRNA Unc13b-206 >lncRNA Atp8b5-201 >retained intron

Gm26881-201 >lncRNA Unc13b-213 >protein coding Unc13b-211 >retained intron

Gm26881-203 >lncRNA Unc13b-216 >protein coding

Unc13b-204 >retained intron Atp8b5-203 >nonsense mediated decay

Unc13b-201 >protein coding

Unc13b-215 >protein coding

Unc13b-210 >lncRNA Atp8b5-205 >nonsense mediated decay

Unc13b-214 >retained intron Unc13b-205 >retained intron

Unc13b-202 >protein coding

Unc13b-212 >protein coding

Unc13b-203 >protein coding

Unc13b-209 >retained intron

Unc13b-207 >retained intron

Atp8b5-204 >protein coding

Atp8b5-202 >protein coding

Unc13b-208 >protein coding

Contigs AL772176.6 > AL732504.23 > Genes < Gm23709-201snoRNA < Gm25010-201snRNA (Comprehensive set...

Regulatory Build

43.05Mb 43.10Mb 43.15Mb 43.20Mb 43.25Mb Reverse strand 225.92 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000107952

204.55 kb Forward strand

Unc13b-202 >protein coding

ENSMUSP00000103... MobiDB lite Low complexity (Seg) Superfamily SSF49562

SSF57889 SMART Protein kinase C-like, phorbol ester/diacylglycerol-binding domain

Calcium-dependent secretion activator domain

C2 domain Prints C2 domain Pfam Calcium-dependent secretion activator domain

Protein kinase C-like, phorbol ester/diacylglycerol-binding domain

C2 domain

Mammalian uncoordinated homology 13, subgroup, domain 2 PROSITE profiles Munc13 homology 1 Mammalian uncoordinated homology 13, domain 2

C2 domain

Protein kinase C-like, phorbol ester/diacylglycerol-binding domain PROSITE patterns Protein kinase C-like, phorbol ester/diacylglycerol-binding domain PANTHER PTHR10480:SF8

Protein Unc-13 Gene3D 3.30.60.20 1.10.357.50 1.20.58.1100

C2 domain superfamily CDD cd08394 Protein Unc-13, C2B domain cd08395

Protein kinase C-like, phorbol ester/diacylglycerol-binding domain

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 1602

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

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