https://www.alphaknockout.com

Mouse Lrrn3 Knockout Project (CRISPR/Cas9)

Objective: To create a Lrrn3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Lrrn3 (NCBI Reference Sequence: NM_010733 ; Ensembl: ENSMUSG00000036295 ) is located on Mouse 12. 2 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000043884). Exon 2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous null mutant mice exhibited increased mean percent body fat and male homozygous mutant mice exhibited enhanced glucose tolerance when compared with controls.

Exon 2 starts from about 0.05% of the coding region. Exon 2 covers 100.0% of the coding region. The size of effective KO region: ~2119 bp. The KO 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

Legends Exon of mouse Lrrn3 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of start codon 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of stop codon 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(35.15% 703) | C(17.85% 357) | T(29.65% 593) | G(17.35% 347)

Note: The 2000 bp section upstream of start codon 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(36.75% 735) | C(15.3% 306) | T(32.65% 653) | G(15.3% 306)

Note: The 2000 bp section downstream of stop codon 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 2000 1 2000 2000 100.0% chr12 - 41454317 41456316 2000 browser details YourSeq 23 1618 1643 2000 83.4% chr17 + 4216808 4216831 24 browser details YourSeq 22 660 685 2000 92.4% chr6 - 97926026 97926051 26 browser details YourSeq 20 488 507 2000 100.0% chr3 - 155642099 155642118 20

Note: The 2000 bp section upstream of start codon 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 2000 1 2000 2000 100.0% chr12 - 41450196 41452195 2000 browser details YourSeq 30 1196 1238 2000 85.8% chr11 - 22924856 22924916 61 browser details YourSeq 29 1207 1243 2000 89.2% chr5 + 137093221 137093257 37 browser details YourSeq 29 1205 1241 2000 89.2% chr16 + 32355465 32355501 37 browser details YourSeq 28 1205 1240 2000 88.9% chr1 - 13377261 13377296 36 browser details YourSeq 28 977 1026 2000 96.7% chr3 + 62981568 62981619 52 browser details YourSeq 27 1205 1239 2000 88.6% chr1 - 58655510 58655544 35 browser details YourSeq 27 1205 1237 2000 91.0% chr3 + 54804299 54804331 33 browser details YourSeq 27 1205 1239 2000 88.6% chr18 + 38046659 38046693 35 browser details YourSeq 26 1205 1238 2000 88.3% chr2 - 131097423 131097456 34 browser details YourSeq 24 1208 1239 2000 87.5% chr19 - 41888835 41888866 32 browser details YourSeq 24 1205 1238 2000 85.3% chr15 - 36217961 36217994 34 browser details YourSeq 23 1208 1240 2000 84.9% chr1 - 183988539 183988571 33 browser details YourSeq 23 1205 1241 2000 81.1% chr3 + 37084571 37084607 37 browser details YourSeq 23 1209 1236 2000 84.0% chr16 + 30504476 30504501 26 browser details YourSeq 23 1205 1237 2000 84.9% chr11 + 65844664 65844696 33 browser details YourSeq 22 1207 1238 2000 84.4% chr16 - 92121731 92121762 32 browser details YourSeq 21 1207 1243 2000 78.4% chr19 + 38250240 38250276 37

Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Lrrn3 leucine rich repeat protein 3, neuronal [ Mus musculus (house mouse) ] Gene ID: 16981, updated on 11-Sep-2019

Gene summary

Official Symbol Lrrn3 provided by MGI Official Full Name leucine rich repeat protein 3, neuronal provided by MGI Primary source MGI:MGI:106036 See related Ensembl:ENSMUSG00000036295 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 NLRR-3 Expression Biased expression in CNS E18 (RPKM 20.4), whole E14.5 (RPKM 13.9) and 6 other tissues See more Orthologs human all

Genomic context

Location: 12; 12 B1 See Lrrn3 in Genome Data Viewer Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (41451668..41486057, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (42178255..42212338, complement)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 1 transcript

Gene: Lrrn3 ENSMUSG00000036295

Description leucine rich repeat protein 3, neuronal [Source:MGI Symbol;Acc:MGI:106036] Gene Synonyms NLRR-3 Location Chromosome 12: 41,451,668-41,486,431 reverse strand. GRCm38:CM001005.2 About this gene This gene has 1 transcript (splice variant), 216 orthologues, 24 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Lrrn3-201 ENSMUST00000043884.5 3865 707aa ENSMUSP00000043818.4 Protein coding CCDS25896 Q8CBC6 TSL:1 GENCODE basic APPRIS P1

54.76 kb Forward strand 41.45Mb 41.46Mb 41.47Mb 41.48Mb 41.49Mb Immp2l-202 >protein coding (Comprehensive set...

Immp2l-203 >protein coding

Contigs AC167364.3 > Genes (Comprehensive set... < Lrrn3-201protein coding

Regulatory Build

41.45Mb 41.46Mb 41.47Mb 41.48Mb 41.49Mb Reverse strand 54.76 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

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Transcript: ENSMUST00000043884

< Lrrn3-201protein coding

Reverse strand 34.76 kb

ENSMUSP00000043... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily SSF52058 type III superfamily

Immunoglobulin-like domain superfamily SMART Leucine-rich repeat N-terminal domain Immunoglobulin subtype 2

Leucine-rich repeat, typical subtype Immunoglobulin subtype

SM00365 Cysteine-rich flanking region, C-terminal Pfam Leucine-rich repeat Fibronectin type III

Immunoglobulin I-set PROSITE profiles Leucine-rich repeat Fibronectin type III

Immunoglobulin-like domain PANTHER PTHR24373:SF7

PTHR24373 Gene3D Leucine-rich repeat domain superfamily Immunoglobulin-like fold CDD Fibronectin type III

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 707

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

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