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Mouse Lmod3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Lmod3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Lmod3 gene (NCBI Reference Sequence: NM_001081157 ; Ensembl: ENSMUSG00000044086 ) is located on Mouse chromosome 6. 3 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 3 (Transcript: ENSMUST00000095655). 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 Lmod3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-427L8 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 an endonuclease-mediated mutation are runted and exhibit nemaline myopathy including a reduction in skeletal myofiber size, centrally nucleated skeletal muscle fibers, increase in skeletal muscle glycogen levels, and abnormal sarcomere and Z lines.

Exon 2 is not frameshift exon, and covers 81.44% of the coding region. The size of intron 1 for 5'-loxP site insertion: 3713 bp, and the size of intron 2 for 3'-loxP site insertion: 8298 bp. The size of effective cKO region: ~1895 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 2 3 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Lmod3 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8395bp) | A(28.72% 2411) | C(20.61% 1730) | T(27.84% 2337) | G(22.84% 1917)

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% chr6 - 97248815 97251814 3000 browser details YourSeq 223 2373 2919 3000 95.9% chr14 + 55695817 55696470 654 browser details YourSeq 192 2403 2936 3000 89.5% chr10 + 108566014 108566453 440 browser details YourSeq 189 2403 2919 3000 87.9% chr6 + 122642578 122642883 306 browser details YourSeq 187 2409 2919 3000 88.6% chr15 - 100549307 100549753 447 browser details YourSeq 184 2709 2919 3000 95.1% chr5 - 129634621 129634925 305 browser details YourSeq 183 2728 2929 3000 96.5% chr9 + 110127176 110127390 215 browser details YourSeq 181 2727 2928 3000 96.0% chr4 - 55306249 55306456 208 browser details YourSeq 181 2739 2945 3000 93.7% chr9 + 53601079 53601272 194 browser details YourSeq 179 2737 2926 3000 97.9% chr13 - 28803463 28803653 191 browser details YourSeq 179 2716 2919 3000 93.7% chr10 + 62927824 62928016 193 browser details YourSeq 178 2391 2912 3000 87.6% chr15 - 90695998 90696261 264 browser details YourSeq 178 2739 2939 3000 96.9% chr13 + 47268886 47269096 211 browser details YourSeq 177 2737 2955 3000 94.0% chr1 - 191553157 191553470 314 browser details YourSeq 177 2737 2937 3000 93.3% chr8 + 41308442 41308636 195 browser details YourSeq 176 2737 2935 3000 95.3% chr4 - 125233616 125233813 198 browser details YourSeq 176 2737 2919 3000 98.4% chr7 + 116354268 116354452 185 browser details YourSeq 176 2737 2919 3000 98.4% chr2 + 69881527 69881713 187 browser details YourSeq 175 2738 2919 3000 98.4% chr5 - 142362671 142362854 184 browser details YourSeq 175 2719 2919 3000 94.2% chr15 - 51886096 51886292 197

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% chr6 - 97243920 97246919 3000 browser details YourSeq 116 2408 2744 3000 90.9% chr17 - 43213579 43213992 414 browser details YourSeq 113 2461 2685 3000 87.4% chr5 + 112613932 112614177 246 browser details YourSeq 111 2403 2680 3000 85.4% chr5 - 144934858 144935296 439 browser details YourSeq 109 2467 2685 3000 81.8% chr12 - 27762652 27762860 209 browser details YourSeq 106 2467 2687 3000 80.2% chr12 + 85543196 85543388 193 browser details YourSeq 101 2468 2702 3000 86.7% chr1 + 93311174 93311407 234 browser details YourSeq 100 2462 2630 3000 83.9% chr11 - 119236577 119236743 167 browser details YourSeq 99 2490 2654 3000 88.4% chr8 - 14877065 14938842 61778 browser details YourSeq 99 2466 2681 3000 80.5% chr14 + 52352604 52352803 200 browser details YourSeq 98 2543 2783 3000 90.4% chr5 - 117880375 117881080 706 browser details YourSeq 93 2466 2657 3000 79.4% chr7 + 66273285 66273454 170 browser details YourSeq 92 2468 2605 3000 83.9% chr11 + 65539025 65539155 131 browser details YourSeq 90 2471 2654 3000 79.7% chr7 + 67429669 67429836 168 browser details YourSeq 84 2475 2630 3000 80.2% chr2 - 164707039 164707183 145 browser details YourSeq 84 2468 2646 3000 84.0% chr8 + 91757530 91757705 176 browser details YourSeq 83 2491 2604 3000 84.6% chr1 + 130060824 130060929 106 browser details YourSeq 82 2491 2644 3000 75.8% chr1 - 152025473 152025618 146 browser details YourSeq 79 2490 2635 3000 74.5% chr7 - 69020487 69020624 138 browser details YourSeq 79 2498 2685 3000 83.8% chr7 - 4064916 4065105 190

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: Lmod3 leiomodin 3 (fetal) [ Mus musculus (house mouse) ] Gene ID: 320502, updated on 12-Aug-2019

Gene summary

Official Symbol Lmod3 provided by MGI Official Full Name leiomodin 3 (fetal) provided by MGI Primary source MGI:MGI:2444169 See related Ensembl:ENSMUSG00000044086 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 5430424A14Rik Expression Biased expression in heart adult (RPKM 11.2), limb E14.5 (RPKM 3.2) and 1 other tissueS ee more Orthologs human all

Genomic context

Location: 6; 6 D3 See Lmod3 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (97238530..97253217, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (97188522..97202774, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 1 transcript

Gene: Lmod3 ENSMUSG00000044086

Description leiomodin 3 (fetal) [Source:MGI Symbol;Acc:MGI:2444169] Gene Synonyms 5430424A14Rik Location Chromosome 6: 97,238,534-97,252,759 reverse strand. GRCm38:CM000999.2 About this gene This gene has 1 transcript (splice variant), 181 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 21 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Lmod3-201 ENSMUST00000095655.3 2215 571aa ENSMUSP00000093315.2 Protein coding CCDS39576 E9QA62 TSL:1 GENCODE basic APPRIS P1

34.23 kb Forward strand

97.23Mb 97.24Mb 97.25Mb 97.26Mb Genes Arl6ip5-201 >protein coding (Comprehensive set...

Contigs AC155724.8 >

Genes (Comprehensive set... < Lmod3-201protein coding

Regulatory Build

97.23Mb 97.24Mb 97.25Mb 97.26Mb Reverse strand 34.23 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

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Transcript: ENSMUST00000095655

< Lmod3-201protein coding

Reverse strand 14.23 kb

ENSMUSP00000093... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF52047 Pfam Tropomodulin PANTHER Tropomodulin

Leiomodin-3 Gene3D Leucine-rich repeat domain superfamily

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 571

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

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