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Mouse Clptm1l Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Clptm1l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Clptm1l gene (NCBI Reference Sequence: NM_146047 ; Ensembl: ENSMUSG00000021610 ) is located on Mouse chromosome 13. 17 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000022102). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Clptm1l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-269I17 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 exhibit partial prenatal and neonatal lethality; however, surviving mice are fertile and overtly normal with no significant alterations in the development, maturation and differentiation of B- lymphocytes or production of antibodies by antibody secreting cells.

Exon 3 starts from about 16.33% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1191 bp, and the size of intron 3 for 3'-loxP site insertion: 1308 bp. The size of effective cKO region: ~690 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 3 4 17 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Clptm1l 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(7190bp) | A(24.63% 1771) | C(23.06% 1658) | T(26.9% 1934) | G(25.41% 1827)

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% chr13 + 73602973 73605972 3000 browser details YourSeq 232 9 503 3000 85.6% chr11 + 80869357 80869762 406 browser details YourSeq 229 9 505 3000 83.5% chr3 + 154395364 154395757 394 browser details YourSeq 222 9 505 3000 83.3% chr5 + 44526288 44526693 406 browser details YourSeq 220 9 536 3000 92.4% chr8 + 122135215 122135970 756 browser details YourSeq 215 14 324 3000 91.2% chr4 - 94490198 94490507 310 browser details YourSeq 213 1 509 3000 81.8% chr11 + 103555695 103556101 407 browser details YourSeq 206 9 500 3000 83.9% chr7 - 56271954 56272331 378 browser details YourSeq 205 14 498 3000 83.1% chr15 - 39148264 39148638 375 browser details YourSeq 205 14 506 3000 82.9% chr2 + 161631614 161632020 407 browser details YourSeq 203 9 320 3000 88.8% chr10 + 93005171 93005476 306 browser details YourSeq 200 9 329 3000 89.1% chr9 + 51721512 51721839 328 browser details YourSeq 200 1 324 3000 87.1% chr6 + 119790808 119791136 329 browser details YourSeq 199 23 327 3000 88.1% chr10 + 89368584 89368878 295 browser details YourSeq 198 1 329 3000 87.6% chr18 + 74874669 74875017 349 browser details YourSeq 197 1 506 3000 84.3% chrX + 157867042 157867458 417 browser details YourSeq 195 9 498 3000 80.6% chr2 - 80153668 80154052 385 browser details YourSeq 195 41 505 3000 82.9% chr1 + 139293147 139293519 373 browser details YourSeq 193 9 329 3000 89.2% chr5 - 110220121 110220444 324 browser details YourSeq 193 9 326 3000 88.5% chr1 - 157378473 157378798 326

Note: The 3000 bp section upstream of Exon 3 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% chr13 + 73606663 73609662 3000 browser details YourSeq 32 1804 1841 3000 82.9% chr1 - 55205064 55205098 35 browser details YourSeq 31 1813 1849 3000 94.3% chr4 - 132979763 132979806 44 browser details YourSeq 28 1813 1840 3000 100.0% chr1 - 164967023 164967050 28 browser details YourSeq 27 1813 1841 3000 96.6% chr12 - 3448281 3448309 29 browser details YourSeq 27 2827 2868 3000 96.6% chr3 + 155738554 155738597 44 browser details YourSeq 27 1813 1841 3000 96.6% chr10 + 12929661 12929689 29 browser details YourSeq 26 1813 1840 3000 96.5% chr4 - 16285480 16285507 28 browser details YourSeq 26 1813 1840 3000 96.5% chr9 + 117631686 117631713 28 browser details YourSeq 26 1813 1840 3000 96.5% chr3 + 117911596 117911623 28 browser details YourSeq 25 1818 1848 3000 96.3% chr12 + 92462195 92462226 32 browser details YourSeq 24 43 70 3000 85.2% chr5 + 106261386 106261412 27 browser details YourSeq 23 48 74 3000 96.0% chr7 - 64217158 64217186 29 browser details YourSeq 23 1818 1840 3000 100.0% chr16 + 82334659 82334681 23 browser details YourSeq 22 61 82 3000 100.0% chr4 + 35139886 35139907 22 browser details YourSeq 21 1820 1840 3000 100.0% chr7 - 40344656 40344676 21 browser details YourSeq 21 2592 2612 3000 100.0% chr4 - 84333711 84333731 21 browser details YourSeq 21 1813 1833 3000 100.0% chr11 - 98172591 98172611 21 browser details YourSeq 21 1813 1833 3000 100.0% chr6 + 132431506 132431526 21

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Clptm1l CLPTM1-like [ Mus musculus (house mouse) ] Gene ID: 218335, updated on 12-Aug-2019

Gene summary

Official Symbol Clptm1l provided by MGI Official Full Name CLPTM1-like provided by MGI Primary source MGI:MGI:2442892 See related Ensembl:ENSMUSG00000021610 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 Crr9; C130052I12Rik Expression Ubiquitous expression in genital fat pad adult (RPKM 55.3), ovary adult (RPKM 54.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 13; 13 C1 See Clptm1l in Genome Data Viewer

Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (73604002..73620639)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (73741749..73758053)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 4 transcripts

Gene: Clptm1l ENSMUSG00000021610

Description CLPTM1-like [Source:MGI Symbol;Acc:MGI:2442892] Gene Synonyms C130052I12Rik Location Chromosome 13: 73,604,006-73,620,605 forward strand. GRCm38:CM001006.2 About this gene This gene has 4 transcripts (splice variants), 250 orthologues, 1 paralogue, 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

Clptm1l-201 ENSMUST00000022102.8 2340 539aa ENSMUSP00000022102.7 Protein coding CCDS56894 Q8BXA5 TSL:1 GENCODE basic APPRIS P1

Clptm1l-204 ENSMUST00000222343.1 830 No protein - Retained intron - - TSL:2

Clptm1l-202 ENSMUST00000221130.1 736 No protein - Retained intron - - TSL:3

Clptm1l-203 ENSMUST00000221417.1 334 No protein - Retained intron - - TSL:3

36.60 kb Forward strand 73.60Mb 73.61Mb 73.62Mb 73.63Mb Genes (Comprehensive set... Clptm1l-201 >protein coding Tert-206 >nonsense mediated decay

Clptm1l-203 >retained introCnlptm1l-202 >retained intron Tert-202 >nonsense mediated decay

Clptm1l-204 >retained intron Tert-205 >retained intron

Tert-201 >protein coding

Contigs AC158359.2 > Regulatory Build

73.60Mb 73.61Mb 73.62Mb 73.63Mb Reverse strand 36.60 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000022102

16.60 kb Forward strand

Clptm1l-201 >protein coding

ENSMUSP00000022... Transmembrane heli... Low complexity (Seg) Pfam Cleft lip and palate transmembrane 1 PANTHER Cleft lip and palate transmembrane 1

Cleft lip and palate transmembrane protein 1-like protein

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 539

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

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