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Mouse Mmel1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mmel1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mmel1 gene (NCBI Reference Sequence: NM_013783 ; Ensembl: ENSMUSG00000058183 ) is located on Mouse chromosome 4. 24 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 24 (Transcript: ENSMUST00000163732). Exon 7~9 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mmel1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-219E7 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: Homozygous null mice display impaired male fertility. Female fertility is not affected.

Exon 7 starts from about 21.63% of the coding region. The knockout of Exon 7~9 will result in frameshift of the gene. The size of intron 6 for 5'-loxP site insertion: 1241 bp, and the size of intron 9 for 3'-loxP site insertion: 1410 bp. The size of effective cKO region: ~1771 bp. The cKO 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 5 6 7 8 9 1011 24 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mmel1 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(8271bp) | A(24.36% 2015) | C(27.24% 2253) | T(22.16% 1833) | G(26.24% 2170)

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% chr4 + 154881697 154884696 3000 browser details YourSeq 209 2758 2976 3000 98.7% chr6 - 87832833 87833052 220 browser details YourSeq 206 2757 2977 3000 97.7% chr9 - 110472380 110472603 224 browser details YourSeq 206 2737 2971 3000 96.4% chr10 + 4350877 4351406 530 browser details YourSeq 203 2757 2969 3000 98.6% chr5 - 147362152 147362379 228 browser details YourSeq 203 2757 2969 3000 98.2% chr11 + 110204258 110379024 174767 browser details YourSeq 202 2753 2980 3000 94.8% chr2 - 51187290 51187509 220 browser details YourSeq 202 2761 2969 3000 99.1% chr11 + 53353480 53353692 213 browser details YourSeq 201 2757 2971 3000 97.2% chr11 - 5384020 5588481 204462 browser details YourSeq 200 2757 2970 3000 95.7% chr18 - 67701968 67702176 209 browser details YourSeq 200 2756 2969 3000 95.3% chr15 - 99446462 99446670 209 browser details YourSeq 200 2757 2972 3000 97.2% chr11 - 52091016 52091233 218 browser details YourSeq 200 2763 2969 3000 99.1% chrX + 152808904 152809116 213 browser details YourSeq 199 2757 2969 3000 95.7% chr16 - 38614434 38614641 208 browser details YourSeq 199 2756 2969 3000 94.7% chr12 - 108230733 108230939 207 browser details YourSeq 199 2755 2969 3000 97.2% chr11 - 48773964 48774230 267 browser details YourSeq 199 2765 2985 3000 94.0% chr5 + 145267367 145267583 217 browser details YourSeq 198 2757 2969 3000 98.1% chr2 - 118172316 118172604 289 browser details YourSeq 198 2757 2969 3000 96.6% chr19 - 45759083 45759292 210 browser details YourSeq 198 2757 2970 3000 95.2% chr4 + 44293859 44294067 209

Note: The 3000 bp section upstream of Exon 7 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% chr4 + 154886468 154889467 3000 browser details YourSeq 150 2696 2860 3000 96.4% chr1 - 9763740 9763907 168 browser details YourSeq 150 2711 2921 3000 93.1% chr4 + 41408213 41408801 589 browser details YourSeq 148 2695 2860 3000 95.2% chr1 - 123053234 123053402 169 browser details YourSeq 147 2693 2859 3000 95.2% chr9 + 66582890 66583061 172 browser details YourSeq 145 2693 2860 3000 94.6% chr15 - 50714430 50714597 168 browser details YourSeq 145 2696 2861 3000 95.2% chr2 + 70178594 70178761 168 browser details YourSeq 145 2695 2861 3000 93.5% chr17 + 25125964 25126130 167 browser details YourSeq 145 30 420 3000 92.0% chr12 + 86789007 86789442 436 browser details YourSeq 143 2695 2849 3000 96.8% chr10 - 59245325 59245490 166 browser details YourSeq 143 2705 2859 3000 96.2% chr16 + 90311606 90311760 155 browser details YourSeq 142 2693 2851 3000 93.6% chr2 + 30632905 30633061 157 browser details YourSeq 142 2695 2857 3000 93.8% chr15 + 25770796 25770962 167 browser details YourSeq 141 2701 2860 3000 94.4% chr3 - 157872034 157872193 160 browser details YourSeq 141 2694 2860 3000 92.8% chr1 + 85744369 85744536 168 browser details YourSeq 140 2695 2860 3000 92.7% chr10 - 93306685 93306850 166 browser details YourSeq 139 2700 2860 3000 94.4% chrX + 15601281 15601442 162 browser details YourSeq 139 2695 2860 3000 89.6% chr2 + 45662789 45662951 163 browser details YourSeq 139 2696 2853 3000 94.3% chr12 + 3498539 3498703 165 browser details YourSeq 137 2700 2857 3000 91.7% chr9 - 55597924 55598079 156

Note: The 3000 bp section downstream of Exon 9 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Mmel1 membrane metallo-endopeptidase-like 1 [ Mus musculus (house mouse) ] Gene ID: 27390, updated on 12-Aug-2019

Gene summary

Official Symbol Mmel1 provided by MGI Official Full Name membrane metallo-endopeptidase-like 1 provided by MGI Primary source MGI:MGI:1351603 See related Ensembl:ENSMUSG00000058183 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 NL2; Nl1; SEP; NEP2; NL-1; Mell1; NEPII; NEP2(m) Expression Biased expression in testis adult (RPKM 51.8) and ovary adult (RPKM 4.3) See more Orthologs human all

Genomic context

Location: 4; 4 E2 See Mmel1 in Genome Data Viewer

Exon count: 24

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (154869585..154895530)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (154243694..154269639)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 7 transcripts

Gene: Mmel1 ENSMUSG00000058183

Description membrane metallo-endopeptidase-like 1 [Source:MGI Symbol;Acc:MGI:1351603] Gene Synonyms Mell1, NEPLP alpha, NEPLP beta, NEPLP gamma, Nep2, Nl1, SEP Location Chromosome 4: 154,869,585-154,895,528 forward strand. GRCm38:CM000997.2 About this gene This gene has 7 transcripts (splice variants), 221 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mmel1-207 ENSMUST00000163732.7 2935 766aa ENSMUSP00000131753.1 Protein coding CCDS51401 E9PVK7 TSL:1 GENCODE basic APPRIS P2

Mmel1-201 ENSMUST00000079269.13 2934 768aa ENSMUSP00000078252.7 Protein coding - B1AS15 TSL:5 GENCODE basic APPRIS ALT2

Mmel1-202 ENSMUST00000080559.12 2926 780aa ENSMUSP00000079399.6 Protein coding - E9QPA9 TSL:5 GENCODE basic APPRIS ALT2

Mmel1-203 ENSMUST00000105634.7 2669 782aa ENSMUSP00000101259.1 Protein coding - B1AS17 TSL:5 GENCODE basic APPRIS ALT2

Mmel1-204 ENSMUST00000105635.7 2592 745aa ENSMUSP00000101260.1 Protein coding - B1AS16 TSL:5 GENCODE basic APPRIS ALT2

Mmel1-206 ENSMUST00000131758.1 592 150aa ENSMUSP00000121243.1 Protein coding - B1AS18 CDS 3' incomplete TSL:3

Mmel1-205 ENSMUST00000129623.1 652 No protein - Retained intron - - TSL:3

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45.94 kb Forward strand 154.86Mb 154.87Mb 154.88Mb 154.89Mb 154.90Mb Genes Ttc34-203 >protein coding Mmel1-207 >protein coding (Comprehensive set...

Ttc34-201 >protein coding Mmel1-201 >protein coding

Ttc34-202 >retained intron Mmel1-202 >protein coding

Mmel1-204 >protein coding

Mmel1-203 >protein coding

Mmel1-206 >protein coding Mmel1-205 >retained intron

Contigs AL607032.16 > Genes < Gm13112-201lncRNA < Prxl2b-202lncRNA (Comprehensive set...

< Gm13112-202lncRNA < Prxl2b-201protein coding

< Prxl2b-203protein coding

Regulatory Build

154.86Mb 154.87Mb 154.88Mb 154.89Mb 154.90Mb Reverse strand 45.94 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000163732

25.94 kb Forward strand

Mmel1-207 >protein coding

ENSMUSP00000131... Transmembrane heli... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF55486

Prints Peptidase M13, C-terminal domain Pfam Peptidase M13, N-terminal domain Peptidase M13, C-terminal domain

PANTHER Membrane metallo-endopeptidase-like 1

Peptidase M13 Gene3D Metallopeptidase, catalytic domain superfamily

Peptidase M13, domain 2 CDD Peptidase M13

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

Variant Legend

inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 766

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

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