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Mouse Lrrc41 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Lrrc41 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Lrrc41 gene (NCBI Reference Sequence: NM_153521 ; Ensembl: ENSMUSG00000028703 ) is located on Mouse chromosome 4. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000030471). 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 Lrrc41 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-306K5 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 14.79% 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: 7827 bp, and the size of intron 4 for 3'-loxP site insertion: 3302 bp. The size of effective cKO region: ~1623 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 4 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Lrrc41 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(8123bp) | A(28.15% 2287) | C(23.7% 1925) | T(27.76% 2255) | G(20.39% 1656)

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 + 116085198 116088197 3000 browser details YourSeq 152 1363 1913 3000 90.0% chr7 - 3673370 3673915 546 browser details YourSeq 140 1809 2548 3000 92.4% chr11 + 51633231 51766672 133442 browser details YourSeq 108 1778 1913 3000 94.4% chr10 - 64906045 64906224 180 browser details YourSeq 102 1798 1929 3000 93.4% chr2 - 92339334 92339487 154 browser details YourSeq 101 1805 1929 3000 95.6% chr4 - 136580126 136580272 147 browser details YourSeq 100 1798 1909 3000 95.6% chr4 - 124341593 124341737 145 browser details YourSeq 99 1798 1909 3000 96.4% chr11 + 87161289 87330298 169010 browser details YourSeq 97 1801 1909 3000 95.4% chr1 - 135193205 135193350 146 browser details YourSeq 95 1798 1909 3000 93.7% chrX - 105941884 105942025 142 browser details YourSeq 95 1801 1909 3000 94.5% chr4 - 133168329 133168472 144 browser details YourSeq 95 1798 1909 3000 93.7% chr2 - 166839274 166839419 146 browser details YourSeq 95 1798 1909 3000 92.9% chr11 - 107487994 107488136 143 browser details YourSeq 95 1801 1909 3000 94.5% chr10 - 76088914 76089054 141 browser details YourSeq 95 1800 1909 3000 94.6% chr1 + 77443621 77443764 144 browser details YourSeq 95 1801 1909 3000 94.5% chr1 + 33759859 33759999 141 browser details YourSeq 94 1806 1908 3000 96.2% chr4 - 109285739 109285872 134 browser details YourSeq 94 1798 1909 3000 92.9% chr1 - 55902893 55903036 144 browser details YourSeq 94 1798 1909 3000 92.8% chrX + 39925843 39926003 161 browser details YourSeq 94 1798 1909 3000 92.8% chr5 + 5731711 5731858 148

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% chr4 + 116089821 116092820 3000 browser details YourSeq 140 2763 2933 3000 88.9% chr1 + 183167241 183167403 163 browser details YourSeq 137 2779 2933 3000 94.8% chr2 - 34721864 34722020 157 browser details YourSeq 136 2765 2935 3000 91.6% chr8 - 105625417 105625597 181 browser details YourSeq 136 2647 2926 3000 83.8% chr4 - 41584708 41584875 168 browser details YourSeq 136 2756 2926 3000 90.7% chr2 + 163683515 163683683 169 browser details YourSeq 135 2779 2933 3000 94.2% chr17 + 6291699 6291859 161 browser details YourSeq 133 2779 2933 3000 93.5% chr6 - 115779364 115779518 155 browser details YourSeq 133 2779 2933 3000 93.5% chr6 + 88040750 88040904 155 browser details YourSeq 133 2778 2927 3000 94.7% chr2 + 126715368 126715519 152 browser details YourSeq 132 2779 2934 3000 89.5% chr2 - 160658089 160658240 152 browser details YourSeq 132 2779 2933 3000 90.8% chr4 + 98851147 98851298 152 browser details YourSeq 132 2778 2933 3000 91.6% chr3 + 89154551 89154705 155 browser details YourSeq 132 2781 2935 3000 94.1% chr10 + 69662682 69662836 155 browser details YourSeq 131 2782 2933 3000 90.7% chr18 - 17088057 17088205 149 browser details YourSeq 131 2779 2924 3000 95.3% chr15 - 85133205 85133352 148 browser details YourSeq 131 2762 2926 3000 91.8% chr14 - 78934048 78934218 171 browser details YourSeq 131 2779 2935 3000 92.3% chr13 - 34759785 34759942 158 browser details YourSeq 131 2691 2933 3000 85.4% chr5 + 50701635 50701790 156 browser details YourSeq 130 2778 2928 3000 94.6% chr11 - 82869505 82869666 162

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: Lrrc41 leucine rich repeat containing 41 [ Mus musculus (house mouse) ] Gene ID: 230654, updated on 12-Aug-2019

Gene summary

Official Symbol Lrrc41 provided by MGI Official Full Name leucine rich repeat containing 41 provided by MGI Primary source MGI:MGI:2441984 See related Ensembl:ENSMUSG00000028703 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 MUF1; AA409966; AW555107; D630045E04Rik; D730026A16Rik Expression Ubiquitous expression in ovary adult (RPKM 72.1), testis adult (RPKM 51.2) and 28 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 D1 See Lrrc41 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (116075269..116097109)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (115748070..115769648)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 6 transcripts

Gene: Lrrc41 ENSMUSG00000028703

Description leucine rich repeat containing 41 [Source:MGI Symbol;Acc:MGI:2441984] Gene Synonyms D630045E04Rik, D730026A16Rik, MUF1 Location Chromosome 4: 116,075,269-116,097,043 forward strand. GRCm38:CM000997.2 About this gene This gene has 6 transcripts (splice variants), 168 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Lrrc41-201 ENSMUST00000030471.8 3065 807aa ENSMUSP00000030471.8 Protein coding CCDS84786 Q8K1C9 TSL:1 GENCODE basic APPRIS P1

Lrrc41-206 ENSMUST00000154274.7 584 No protein - lncRNA - - TSL:3

Lrrc41-203 ENSMUST00000134983.1 526 No protein - lncRNA - - TSL:1

Lrrc41-204 ENSMUST00000138952.7 451 No protein - lncRNA - - TSL:3

Lrrc41-202 ENSMUST00000133799.7 353 No protein - lncRNA - - TSL:3

Lrrc41-205 ENSMUST00000152577.1 305 No protein - lncRNA - - TSL:3

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41.77 kb Forward strand 116.07Mb 116.08Mb 116.09Mb 116.10Mb Genes (Comprehensive set... Gm12854-201 >processed pseudogLerrnce41-201 >protein coding

Lrrc41-206 >lncRNA Lrrc41-203 >lncRNA

Lrrc41-202 >lncRNA

Lrrc41-204 >lncRNA

Lrrc41-205 >lncRNA

Contigs AL627105.13 > AL611947.11 >

Genes < Uqcrh-202protein coding < Rad54l-204lncRNA (Comprehensive set...

< Uqcrh-204lncRNA < Rad54l-202protein coding

< Uqcrh-201protein coding < Rad54l-201protein coding

< Uqcrh-205retained intron

< Uqcrh-203retained intron

Regulatory Build

116.07Mb 116.08Mb 116.09Mb 116.10Mb Reverse strand 41.77 kb

Regulation Legend CTCF 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 pseudogene

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Transcript: ENSMUST00000030471

21.77 kb Forward strand

Lrrc41-201 >protein coding

ENSMUSP00000030... MobiDB lite Low complexity (Seg) Superfamily SSF52047 SMART SM00368 PANTHER Leucine-rich repeat-containing 41 Gene3D Leucine-rich repeat domain superfamily

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

Variant Legend inframe insertion missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 807

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

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