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Mouse Lynx1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Lynx1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Lynx1 (NCBI Reference Sequence: NM_011838 ; Ensembl: ENSMUSG00000022594 ) is located on Mouse 15. 4 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000023259). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Lynx1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-388I10 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 exhibit increased sensitivity to nicotine, neurodegeneration, brain vacuoles amd improved cue-conditioned learning.

Exon 2~4 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 4. The size of intron 1 for 5'-loxP site insertion: 847 bp. The size of effective cKO region: ~946 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele T A

5' gRNA region A 3'

1 2 3 4

Targeting vector T A A

Targeted allele T A A

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Lynx1 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(7173bp) | A(25.76% 1848) | C(25.67% 1841) | T(22.68% 1627) | G(25.89% 1857)

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% chr15 - 74752157 74755156 3000 browser details YourSeq 911 1 1021 3000 97.2% chr7 - 142829082 142830355 1274 browser details YourSeq 901 1 956 3000 97.4% chrX - 6613616 6614573 958 browser details YourSeq 900 1 1021 3000 96.5% chr10 - 40046333 40047608 1276 browser details YourSeq 898 2 956 3000 97.1% chr12 + 102848395 102849351 957 browser details YourSeq 896 1 956 3000 97.0% chr3 - 52807830 52808786 957 browser details YourSeq 896 1 1021 3000 97.0% chr11 - 90203077 90204352 1276 browser details YourSeq 894 1 1021 3000 96.2% chr17 + 94788965 94790236 1272 browser details YourSeq 893 1 1021 3000 96.2% chr3 - 67239270 67240547 1278 browser details YourSeq 891 1 956 3000 96.7% chr11 + 23923681 23924638 958 browser details YourSeq 890 1 1021 3000 96.1% chr7 - 3967479 3968755 1277 browser details YourSeq 890 1 1021 3000 96.3% chr1 - 106414082 106415355 1274 browser details YourSeq 890 1 1021 3000 96.2% chrX + 107502085 107503360 1276 browser details YourSeq 890 1 953 3000 97.2% chr14 + 66945690 66946645 956 browser details YourSeq 889 1 956 3000 96.7% chr4 - 62269998 62270956 959 browser details YourSeq 889 1 1021 3000 96.0% chr2 - 103157729 103158989 1261 browser details YourSeq 888 1 956 3000 96.6% chr9 + 115462227 115463182 956 browser details YourSeq 887 1 1021 3000 96.5% chr4 - 48845683 48846970 1288 browser details YourSeq 886 1 956 3000 96.0% chrX - 103102472 103103423 952 browser details YourSeq 886 1 1021 3000 96.0% chr11 - 9101560 9102836 1277

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% chr15 - 74748234 74751233 3000 browser details YourSeq 20 450 469 3000 100.0% chr6 + 32517744 32517763 20

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 information: Lynx1 Ly6/neurotoxin 1 [ Mus musculus (house mouse) ] Gene ID: 23936, updated on 10-Oct-2019

Gene summary

Official Symbol Lynx1 provided by MGI Official Full Name Ly6/neurotoxin 1 provided by MGI Primary source MGI:MGI:1345180 See related Ensembl:ENSMUSG00000022594 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as SLURP-2; AI838844 Expression Biased expression in cerebellum adult (RPKM 74.6), heart adult (RPKM 72.6) and 14 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 D3 See Lynx1 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (74747856..74752979, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (74578286..74583409, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 3 transcripts

Gene: Lynx1 ENSMUSG00000022594

Description Ly6/neurotoxin 1 [Source:MGI Symbol;Acc:MGI:1345180] Location Chromosome 15: 74,747,852-74,753,046 reverse strand. GRCm38:CM001008.2 About this gene This gene has 3 transcripts (splice variants), 77 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 15 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Lynx1-201 ENSMUST00000023259.14 4023 116aa ENSMUSP00000023259.8 Protein coding CCDS27530 P0DP60 TSL:1 GENCODE basic APPRIS P1

Lynx1-202 ENSMUST00000189128.1 436 34aa ENSMUSP00000139494.1 Protein coding - A0A087WNU3 CDS 3' incomplete TSL:3

Lynx1-203 ENSMUST00000189696.1 706 No protein - Retained intron - - TSL:NA

25.20 kb Forward strand 74.74Mb 74.75Mb 74.76Mb Contigs AC118022.12 > (Comprehensive set... < Slurp2-201protein coding < Lynx1-201protein coding < Ly6d-201protein coding

< Lynx1-202protein coding

< Lynx1-203retained intron

Regulatory Build

74.74Mb 74.75Mb 74.76Mb Reverse strand 25.20 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000023259

< Lynx1-201protein coding

Reverse strand 5.20 kb

ENSMUSP00000023... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily SSF57302

SMART Ly-6 antigen/uPA receptor-like

Pfam Snake toxin/toxin-like PANTHER PTHR16983:SF27

PTHR16983 Gene3D 2.10.60.10 CDD cd00117

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

Variant Legend

synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 100 116

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

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