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Mouse Lmbr1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Lmbr1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Lmbr1 (NCBI Reference Sequence: NM_020295 ; Ensembl: ENSMUSG00000010721 ) is located on Mouse 5. 17 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000055195). Exon 6~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Lmbr1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-221J6 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 null allele show minor coalitions of distal wrist bones and a low incidence of limb defects, including oligodactyly, brachyphalangia, and soft tissue or bony syndactyly. Homozygotes for another null allele exhibit normal morphology,clinical chemistry, hematology and behavior.

Exon 6 starts from about 28.84% of the coding region. The knockout of Exon 6~7 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 30852 bp, and the size of intron 7 for 3'-loxP site insertion: 814 bp. The size of effective cKO region: ~1298 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 6 7 8 17 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Lmbr1 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(7798bp) | A(24.66% 1923) | C(19.93% 1554) | T(35.18% 2743) | G(20.24% 1578)

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% chr5 - 29293200 29296199 3000 browser details YourSeq 680 429 1580 3000 93.5% chr15 - 34733559 35383038 649480 browser details YourSeq 678 96 863 3000 95.1% chr14 - 45096361 45097286 926 browser details YourSeq 668 13 866 3000 96.1% chr2 + 126686390 126687247 858 browser details YourSeq 647 429 1567 3000 93.6% chr2 - 68671695 69095425 423731 browser details YourSeq 624 158 866 3000 94.4% chr12 - 74138128 74138857 730 browser details YourSeq 582 157 866 3000 94.8% chr5 + 41275591 41276517 927 browser details YourSeq 455 331 842 3000 94.6% chr14 + 37281506 37282408 903 browser details YourSeq 454 356 866 3000 94.6% chr15 + 5696588 5697249 662 browser details YourSeq 453 1 866 3000 91.3% chr3 + 70887436 70887939 504 browser details YourSeq 440 1 866 3000 91.7% chr8 - 73165633 73166221 589 browser details YourSeq 410 1 866 3000 91.5% chr10 - 3797485 3798012 528 browser details YourSeq 406 429 866 3000 96.6% chr3 + 27009988 27010427 440 browser details YourSeq 402 429 866 3000 95.9% chr6 + 148321002 148321439 438 browser details YourSeq 402 429 866 3000 95.9% chr3 + 10079489 10079926 438 browser details YourSeq 401 429 864 3000 96.2% chr7 - 50276752 50277188 437 browser details YourSeq 399 429 866 3000 95.7% chr4 - 110414261 110414699 439 browser details YourSeq 399 429 866 3000 95.7% chr15 - 31791844 31792282 439 browser details YourSeq 399 429 866 3000 95.7% chr14 - 52536957 52537395 439 browser details YourSeq 399 429 866 3000 95.7% chr1 + 88011627 88012064 438

Note: The 3000 bp section upstream of Exon 6 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% chr5 - 29288902 29291901 3000 browser details YourSeq 38 396 467 3000 85.2% chr5 + 5363306 5363375 70 browser details YourSeq 26 506 537 3000 96.5% chr7 - 34673798 34673831 34 browser details YourSeq 25 22 49 3000 84.7% chr4 + 53544821 53544846 26 browser details YourSeq 24 2274 2298 3000 100.0% chr12 + 9708270 9708296 27 browser details YourSeq 22 1743 1767 3000 95.9% chr15 - 96386561 96386586 26 browser details YourSeq 20 1846 1887 3000 73.9% chr10 - 82785385 82785426 42

Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Lmbr1 limb region 1 [ Mus musculus (house mouse) ] Gene ID: 56873, updated on 12-Aug-2019

Gene summary

Official Symbol Lmbr1 provided by MGI Official Full Name limb region 1 provided by MGI Primary source MGI:MGI:1861746 See related Ensembl:ENSMUSG00000010721 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 AU017641; 1110048D14Rik Summary This gene encodes a member of the LMBR1-like membrane protein family. Another member of this protein family has been Expression shown to be a lipocalin transmembrane receptor. A highly conserved, cis-acting regulatory module for the gene is located within an intron of this gene. Consequently, disruption of this genic region can alter sonic hedgehog expression and affect limb patterning, but it is not known if this gene functions directly in limb development. [provided by RefSeq, Jul 2008] Orthologs Ubiquitous expression in testis adult (RPKM 2.4), CNS E18 (RPKM 2.2) and 28 other tissues See more human all

Genomic context

Location: 5 B1; 5 14.81 cM See Lmbr1 in Genome Data Viewer

Exon count: 21

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (29229802..29378421, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (29556342..29704930, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 7 transcripts

Gene: Lmbr1 ENSMUSG00000010721

Description limb region 1 [Source:MGI Symbol;Acc:MGI:1861746] Gene Synonyms 1110048D14Rik Location Chromosome 5: 29,229,802-29,378,390 reverse strand. GRCm38:CM000998.2 About this gene This gene has 7 transcripts (splice variants), 197 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Lmbr1- ENSMUST00000055195.10 4926 490aa ENSMUSP00000058405.4 Protein coding CCDS19148 Q9JIT0 TSL:1 201 GENCODE basic APPRIS P1

Lmbr1- ENSMUST00000179191.5 4836 462aa ENSMUSP00000136160.1 Protein coding - J3QM78 TSL:5 202 GENCODE basic

Lmbr1- ENSMUST00000196321.4 3422 367aa ENSMUSP00000143348.1 Protein coding - A0A0G2JFX9 TSL:1 203 GENCODE basic

Lmbr1- ENSMUST00000198105.4 2274 463aa ENSMUSP00000142755.1 Protein coding - Q9JIT0 TSL:1 204 GENCODE basic

Lmbr1- ENSMUST00000200564.4 1598 313aa ENSMUSP00000143316.1 Protein coding - A0A0G2JFU9 TSL:5 207 GENCODE basic

Lmbr1- ENSMUST00000200149.1 714 110aa ENSMUSP00000142987.1 Nonsense mediated - A0A0G2JF18 CDS 5' 206 decay incomplete TSL:3

Lmbr1- ENSMUST00000198367.1 734 No - lncRNA - - TSL:3 205 protein

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168.59 kb Forward strand 29.22Mb 29.24Mb 29.26Mb 29.28Mb 29.30Mb 29.32Mb 29.34Mb 29.36Mb 29.38Mb Rnf32-205 >nonsense mediated decay 4632411P08Rik-201 >lncRNA (Comprehensive set...

Rnf32-201 >protein coding

Rnf32-204 >protein coding

Rnf32-211 >protein coding

Rnf32-209 >protein coding

Rnf32-210 >retained intron

Contigs AC159298.3 > AC175116.2 > AC171500.2 > Genes (Comprehensive set... < Lmbr1-201protein coding

< Lmbr1-202protein coding

< Lmbr1-203protein coding

< Lmbr1-204protein coding

< Lmbr1-207protein coding

< Lmbr1-206nonsense mediated decay < Gm23801-201miRNA

< Lmbr1-205lncRNA

< C79130-201TEC

Regulatory Build

29.22Mb 29.24Mb 29.26Mb 29.28Mb 29.30Mb 29.32Mb 29.34Mb 29.36Mb 29.38Mb Reverse strand 168.59 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: ENSMUST00000055195

< Lmbr1-201protein coding

Reverse strand 148.58 kb

ENSMUSP00000058... Transmembrane heli... Low complexity (Seg) Coiled-coils (Ncoils) Prints Lipocalin-interacting membrane receptor Pfam LMBR1-like membrane protein PANTHER Lipocalin-interacting membrane receptor

PTHR12625:SF1

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 490

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

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