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Mouse Lancl2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Lancl2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Lancl2 gene (NCBI Reference Sequence: NM_133737 ; Ensembl: ENSMUSG00000062190 ) is located on Mouse chromosome 6. 9 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000050077). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Lancl2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-291E6 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 3 starts from about 23.93% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 9391 bp, and the size of intron 3 for 3'-loxP site insertion: 784 bp. The size of effective cKO region: ~1306 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 3 4 5 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Lancl2 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(7208bp) | A(25.72% 1854) | C(17.18% 1238) | T(35.86% 2585) | G(21.24% 1531)

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% chr6 + 57719248 57722247 3000 browser details YourSeq 598 6 1142 3000 85.8% chr4 - 140893520 140894708 1189 browser details YourSeq 561 370 1672 3000 87.0% chr2 - 18404264 18405603 1340 browser details YourSeq 507 12 1159 3000 81.6% chr10 + 129643202 130193039 549838 browser details YourSeq 492 5 1063 3000 84.0% chr2 - 102490681 102491798 1118 browser details YourSeq 489 91 1127 3000 83.9% chr13 - 74239264 74240355 1092 browser details YourSeq 489 8 1066 3000 84.5% chr13 - 65082782 65084048 1267 browser details YourSeq 485 386 1603 3000 86.1% chr2 - 133607275 133608603 1329 browser details YourSeq 484 520 2059 3000 84.7% chr6 - 96950436 96952164 1729 browser details YourSeq 482 549 1702 3000 84.9% chr5 - 30080898 30082092 1195 browser details YourSeq 480 5 977 3000 85.9% chr4 - 42907843 42908827 985 browser details YourSeq 472 5 796 3000 83.8% chr1 - 38112344 38113194 851 browser details YourSeq 469 6 1071 3000 84.8% chr1 - 6111084 6112170 1087 browser details YourSeq 464 238 1146 3000 83.6% chr13 + 91712712 91713686 975 browser details YourSeq 457 91 1331 3000 82.0% chr12 - 113389610 113392876 3267 browser details YourSeq 452 13 862 3000 81.7% chr14 + 31678232 31679116 885 browser details YourSeq 445 5 1005 3000 82.1% chr1 + 172772036 172773354 1319 browser details YourSeq 430 5 1020 3000 78.3% chr12 - 64578235 64579278 1044 browser details YourSeq 427 58 1068 3000 83.8% chr1 - 56473793 56474800 1008 browser details YourSeq 423 8 1021 3000 83.8% chr9 + 115878330 115879437 1108

Note: The 3000 bp section upstream of Exon 3 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% chr6 + 57722956 57725955 3000 browser details YourSeq 120 2840 2997 3000 91.8% chr7 + 92873026 92873187 162 browser details YourSeq 117 2840 2997 3000 85.8% chr13 - 35933070 35933220 151 browser details YourSeq 116 2840 3000 3000 84.1% chr6 - 18854396 18854540 145 browser details YourSeq 113 2840 2980 3000 90.7% chr8 - 110442992 110443141 150 browser details YourSeq 113 2838 2997 3000 83.0% chr1 + 145773921 145774069 149 browser details YourSeq 112 2840 2997 3000 85.4% chr15 - 44772022 44772175 154 browser details YourSeq 111 2839 2996 3000 82.4% chr12 - 65214109 65214256 148 browser details YourSeq 111 2840 2997 3000 84.8% chr10 - 113086295 113086445 151 browser details YourSeq 111 2840 2997 3000 82.5% chr1 - 12086623 12086772 150 browser details YourSeq 111 2840 2992 3000 84.6% chr10 + 25217472 25217610 139 browser details YourSeq 110 2840 2994 3000 85.2% chr12 - 31880936 31881088 153 browser details YourSeq 109 2840 2992 3000 83.5% chr17 - 92709436 92709575 140 browser details YourSeq 109 2840 2996 3000 83.3% chr15 - 71435816 71435955 140 browser details YourSeq 109 2837 2989 3000 84.1% chr1 + 58460069 58460215 147 browser details YourSeq 107 2829 2977 3000 88.7% chr1 - 156923951 156924397 447 browser details YourSeq 107 2840 2997 3000 83.3% chr16 + 21435985 21436117 133 browser details YourSeq 106 2850 2997 3000 86.5% chr8 - 53771416 53771561 146 browser details YourSeq 106 2840 2997 3000 83.5% chr17 - 43682362 43682505 144 browser details YourSeq 105 2840 2982 3000 86.8% chr9 - 48855749 48855891 143

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Lancl2 LanC (bacterial lantibiotic synthetase component C)-like 2 [ Mus musculus (house mouse) ] Gene ID: 71835, updated on 10-Oct-2019

Gene summary

Official Symbol Lancl2 provided by MGI Official Full Name LanC (bacterial lantibiotic synthetase component C)-like 2 provided by MGI Primary source MGI:MGI:1919085 See related Ensembl:ENSMUSG00000062190 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 1700003F10Rik Expression Broad expression in testis adult (RPKM 25.7), CNS E18 (RPKM 18.4) and 18 other tissues See more Orthologs human all

Genomic context

Location: 6; 6 B3 See Lancl2 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (57702455..57739449)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (57652449..57689443)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 3 transcripts

Gene: Lancl2 ENSMUSG00000062190

Description LanC (bacterial lantibiotic synthetase component C)-like 2 [Source:MGI Symbol;Acc:MGI:1919085] Gene Synonyms 1700003F10Rik Location Chromosome 6: 57,702,540-57,739,439 forward strand. GRCm38:CM000999.2 About this gene This gene has 3 transcripts (splice variants), 184 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Lancl2- ENSMUST00000050077.14 2509 450aa ENSMUSP00000052146.8 Protein coding CCDS20185 Q9JJK2 TSL:1 201 GENCODE basic APPRIS P1

Lancl2- ENSMUST00000072954.7 2245 450aa ENSMUSP00000072723.1 Protein coding CCDS20185 Q9JJK2 TSL:1 202 GENCODE basic APPRIS P1

Lancl2- ENSMUST00000153777.1 1948 441aa ENSMUSP00000121377.1 Nonsense mediated - F6RJV6 CDS 5' 203 decay incomplete TSL:1

56.90 kb Forward strand

57.70Mb 57.71Mb 57.72Mb 57.73Mb 57.74Mb Genes (Comprehensive set... Lancl2-202 >protein coding

Lancl2-201 >protein coding

Lancl2-203 >nonsense mediated decay

Contigs AC127307.3 > Genes < Vopp1-202protein coding (Comprehensive set...

Regulatory Build

57.70Mb 57.71Mb 57.72Mb 57.73Mb 57.74Mb Reverse strand 56.90 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000050077

35.85 kb Forward strand

Lancl2-201 >protein coding

ENSMUSP00000052... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF158745 SMART Lanthionine synthetase C-like Prints LanC-like protein, eukaryotic

Lanthionine synthetase C-like Pfam Lanthionine synthetase C-like PANTHER PTHR12736:SF11

PTHR12736 Gene3D Six-hairpin glycosidase-like superfamily CDD LanC-like protein, eukaryotic

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 450

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

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