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Mouse Tmem216 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tmem216 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tmem216 gene (NCBI Reference Sequence: NM_026798 ; Ensembl: ENSMUSG00000024667 ) is located on Mouse chromosome 19. 5 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000025569). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tmem216 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-29G10 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:

Exon 4 starts from about 18.01% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2554 bp, and the size of intron 4 for 3'-loxP site insertion: 732 bp. The size of effective cKO region: ~702 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 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tmem216 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(7202bp) | A(26.83% 1932) | C(22.23% 1601) | T(29.8% 2146) | G(21.15% 1523)

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% chr19 - 10552225 10555224 3000 browser details YourSeq 189 2448 2983 3000 89.9% chr13 - 100558433 100558969 537 browser details YourSeq 174 2434 2841 3000 82.6% chr6 + 113274045 113274352 308 browser details YourSeq 165 2445 2839 3000 89.1% chr1 + 160375723 160376232 510 browser details YourSeq 162 2653 2901 3000 91.3% chr2 + 65901937 65902228 292 browser details YourSeq 162 2434 2842 3000 87.1% chr15 + 89200416 89200916 501 browser details YourSeq 161 2405 2972 3000 89.2% chr10 + 76431756 76432441 686 browser details YourSeq 160 2415 2843 3000 87.5% chr4 - 45634927 45635350 424 browser details YourSeq 160 2406 2845 3000 84.0% chr12 - 70509318 70509525 208 browser details YourSeq 160 2352 2846 3000 82.7% chr1 + 58639670 58639926 257 browser details YourSeq 159 2406 2845 3000 89.9% chr13 - 51586546 51587071 526 browser details YourSeq 158 2416 2841 3000 89.6% chr5 + 120604095 120604588 494 browser details YourSeq 152 2661 2846 3000 88.3% chr7 - 116309106 116309283 178 browser details YourSeq 149 2664 2845 3000 88.8% chr8 - 80690226 80690403 178 browser details YourSeq 149 2667 2844 3000 90.2% chr6 - 38331094 38331267 174 browser details YourSeq 149 2654 2843 3000 90.8% chr14 - 70614058 70614249 192 browser details YourSeq 149 2653 2843 3000 91.7% chr11 - 95110677 95110879 203 browser details YourSeq 149 2661 2845 3000 89.6% chr12 + 83925794 83925976 183 browser details YourSeq 148 2659 2843 3000 88.4% chr2 - 31868121 31868301 181 browser details YourSeq 147 2661 2845 3000 89.0% chr19 - 31110132 31110314 183

Note: The 3000 bp section upstream of Exon 4 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% chr19 - 10548523 10551522 3000 browser details YourSeq 103 257 555 3000 87.1% chr11 + 115742278 115742715 438 browser details YourSeq 101 263 434 3000 83.0% chr10 + 63103572 63103742 171 browser details YourSeq 98 260 438 3000 89.6% chr4 + 44020770 44021260 491 browser details YourSeq 93 262 442 3000 86.2% chr1 + 86329512 86329706 195 browser details YourSeq 91 256 440 3000 79.4% chr10 - 91166200 91166362 163 browser details YourSeq 90 297 439 3000 80.8% chr10 - 78089018 78089157 140 browser details YourSeq 90 298 460 3000 78.1% chr11 + 87058363 87058522 160 browser details YourSeq 89 300 438 3000 81.9% chr7 - 99518606 99518741 136 browser details YourSeq 89 296 440 3000 80.0% chr14 - 54415006 54415147 142 browser details YourSeq 88 298 439 3000 80.2% chr9 - 73129218 73129344 127 browser details YourSeq 88 295 448 3000 83.4% chr4 - 133834116 133834279 164 browser details YourSeq 88 297 440 3000 80.4% chr7 + 101045409 101045549 141 browser details YourSeq 86 299 440 3000 80.0% chrX - 41927660 41927798 139 browser details YourSeq 85 296 440 3000 79.0% chr2 + 60266476 60266617 142 browser details YourSeq 85 296 441 3000 89.8% chr15 + 36061054 36061200 147 browser details YourSeq 84 298 458 3000 74.5% chr4 - 124697127 124697274 148 browser details YourSeq 84 296 431 3000 80.2% chr11 + 80245133 80245265 133 browser details YourSeq 82 300 440 3000 79.1% chr7 + 97561896 97562032 137 browser details YourSeq 81 296 438 3000 84.5% chr11 + 97430524 97430666 143

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tmem216 transmembrane protein 216 [ Mus musculus (house mouse) ] Gene ID: 68642, updated on 12-Aug-2019

Gene summary

Official Symbol Tmem216 provided by MGI Official Full Name transmembrane protein 216 provided by MGI Primary source MGI:MGI:1920020 See related Ensembl:ENSMUSG00000024667 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 Also known as AI482550; 1110017C22Rik; 2810441K11Rik; 4921533J23Rik; A930021F15Rik Summary This gene encodes a transmembrane protein which is involved in regulation of signaling and trafficking of associated Expression proteins. In humans, mutations in this gene are associated with ciliopathies including Joubert, Meckel and related syndromes. Alternative splicing of this gene results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Apr 2013] Orthologs Ubiquitous expression in CNS E14 (RPKM 13.3), whole brain E14.5 (RPKM 12.9) and 28 other tissues See more human all

Genomic context

Location: 19; 19 A See Tmem216 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (10539326..10556297, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (10624956..10630728, complement)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 8 transcripts

Gene: Tmem216 ENSMUSG00000024667

Description transmembrane protein 216 [Source:MGI Symbol;Acc:MGI:1920020] Gene Synonyms 1110017C22Rik, 2810441K11Rik, 4921533J23Rik, A930021F15Rik Location Chromosome 19: 10,533,865-10,556,238 reverse strand. GRCm38:CM001012.2 About this gene This gene has 8 transcripts (splice variants), 193 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tmem216- ENSMUST00000025569.8 1101 87aa ENSMUSP00000025569.2 Protein coding CCDS37913 Q9CQC4 TSL:1 201 GENCODE basic

Tmem216- ENSMUST00000059582.8 999 87aa ENSMUSP00000059878.2 Protein coding CCDS37913 Q9CQC4 TSL:1 202 GENCODE basic

Tmem216- ENSMUST00000154383.1 927 141aa ENSMUSP00000115319.1 Protein coding CCDS70934 Q9CQC4 TSL:3 206 GENCODE basic APPRIS P1

Tmem216- ENSMUST00000123788.7 935 141aa ENSMUSP00000119596.1 Nonsense mediated - Q9CQC4 TSL:3 203 decay

Tmem216- ENSMUST00000236561.1 876 87aa ENSMUSP00000158207.1 Nonsense mediated - Q9CQC4 - 207 decay

Tmem216- ENSMUST00000145210.7 774 87aa ENSMUSP00000123397.1 Nonsense mediated - Q9CQC4 TSL:1 205 decay

Tmem216- ENSMUST00000237507.1 1848 No - Retained intron - - - 208 protein

Tmem216- ENSMUST00000131205.1 471 No - Retained intron - - TSL:3 204 protein

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42.37 kb Forward strand 10.53Mb 10.54Mb 10.55Mb 10.56Mb Genes Cpsf7-201 >protein coding (Comprehensive set...

Cpsf7-205 >nonsense mediated decay

Cpsf7-207 >protein coding

Cpsf7-202 >retained intron

Cpsf7-203 >protein coding

Cpsf7-206 >nonsense mediated decay

Cpsf7-208 >nonsense mediated decay

Cpsf7-204 >retained intron

Contigs < AC125093.4 Genes (Comprehensive set... < Sdhaf2-201protein coding < Tmem216-205nonsense mediated decay

< Sdhaf2-206nonsense mediated decay < Tmem216-203nonsense mediated decay

< Sdhaf2-203nonsense mediated decay < Tmem216-201protein coding

< Sdhaf2-204retained intron < Tmem216-207nonsense mediated decay

< Sdhaf2-208protein coding < Tmem216-202protein coding

< Sdhaf2-202protein coding < Tmem216-206protein coding

< Sdhaf2-209lncRNA < Tmem216-208retained intron

< Sdhaf2-207protein coding < Tmem216-204retained intron

Regulatory Build

10.53Mb 10.54Mb 10.55Mb 10.56Mb Reverse strand 42.37 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000025569

< Tmem216-201protein coding

Reverse strand 5.78 kb

ENSMUSP00000025... Transmembrane heli... Pfam Uncharacterised protein family, transmembrane-17

PANTHER Uncharacterised protein family, transmembrane-17

PTHR13531:SF5

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

Variant Legend

synonymous variant

Scale bar 0 8 16 24 32 40 48 56 64 72 87

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

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