https://www.alphaknockout.com

Mouse Lynx1 Knockout Project (CRISPR/Cas9)

Objective: To create a Lynx1 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 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: Mice homozygous for a null allele exhibit increased sensitivity to nicotine, neurodegeneration, brain vacuoles amd improved cue-conditioned learning.

Exon 2 starts from about 0.29% of the coding region. Exon 2~4 covers 100.0% of the coding region. The size of effective KO region: ~671 bp. The KO 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 2 3 4

Legends Exon of mouse Lynx1 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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(20.95% 419) | C(26.85% 537) | T(25.15% 503) | G(27.05% 541)

Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

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

Sequence 12

Summary: Full Length(2000bp) | A(23.65% 473) | C(27.05% 541) | T(20.65% 413) | G(28.65% 573)

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% chr15 - 74751907 74753906 2000 browser details YourSeq 91 79 224 2000 81.5% chr1 - 16093834 16093958 125 browser details YourSeq 90 79 225 2000 78.6% chr10 - 24011209 24011343 135 browser details YourSeq 89 80 224 2000 82.5% chr3 - 102821876 102822000 125 browser details YourSeq 87 105 225 2000 91.6% chr4 + 149681201 149681772 572 browser details YourSeq 86 93 224 2000 81.3% chr9 - 55144717 55144837 121 browser details YourSeq 85 112 225 2000 88.8% chr2 - 32263824 32263934 111 browser details YourSeq 85 108 225 2000 86.5% chr18 + 22471976 22472088 113 browser details YourSeq 84 92 222 2000 82.0% chr12 + 88219145 88219261 117 browser details YourSeq 83 117 223 2000 89.5% chr2 - 131144096 131144451 356 browser details YourSeq 83 93 224 2000 83.4% chr1 - 31428615 31428734 120 browser details YourSeq 83 99 216 2000 89.5% chr2 + 152454146 152454264 119 browser details YourSeq 81 112 223 2000 89.6% chr12 - 21438627 21438737 111 browser details YourSeq 81 112 223 2000 86.6% chr11 - 79260921 79261028 108 browser details YourSeq 80 108 225 2000 84.1% chr2 - 85794020 85794125 106 browser details YourSeq 80 80 225 2000 78.6% chr8 + 120472959 120473072 114 browser details YourSeq 80 112 225 2000 83.9% chr6 + 124395834 124395941 108 browser details YourSeq 79 117 225 2000 85.2% chr5 - 129672613 129672715 103 browser details YourSeq 79 108 225 2000 81.1% chr3 - 110170345 110170457 113 browser details YourSeq 79 112 221 2000 86.2% chr16 - 32441910 32442014 105

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% chr15 - 74749234 74751233 2000 browser details YourSeq 27 621 648 2000 100.0% chr12 + 87025092 87025121 30 browser details YourSeq 24 1670 1694 2000 100.0% chr1 + 76095660 76095685 26 browser details YourSeq 21 967 987 2000 100.0% chr8 - 91933811 91933831 21 browser details YourSeq 21 1675 1695 2000 100.0% chr11 - 28586576 28586596 21 browser details YourSeq 21 1675 1695 2000 100.0% chrX + 22726991 22727011 21 browser details YourSeq 21 1675 1695 2000 100.0% chr7 + 48450726 48450746 21 browser details YourSeq 21 1675 1695 2000 100.0% chr3 + 98719881 98719901 21 browser details YourSeq 21 1675 1695 2000 100.0% chr13 + 15794916 15794936 21 browser details YourSeq 20 1517 1536 2000 100.0% chr1 - 86273771 86273790 20 browser details YourSeq 20 450 469 2000 100.0% chr6 + 32517744 32517763 20

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: 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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