https://www.alphaknockout.com

Mouse Mettl8 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mettl8 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mettl8 gene (NCBI Reference Sequence: NM_145524 ; Ensembl: ENSMUSG00000041975 ) is located on Mouse chromosome 2. 10 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000112186). 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 Mettl8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-88G11 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 knock-out allele exhibit reduced 3-methylcytidine (m3C) methyltransferases modification of mRNA.

Exon 3 starts from about 12.37% 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: 24644 bp, and the size of intron 3 for 3'-loxP site insertion: 11288 bp. The size of effective cKO region: ~1361 bp. The cKO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mettl8 Homology arm cKO region loxP site

Page 2 of 8 https://www.alphaknockout.com

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(7092bp) | A(26.95% 1911) | C(22.87% 1622) | T(28.12% 1994) | G(22.07% 1565)

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.

Page 3 of 8 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr2 - 70993826 70996825 3000 browser details YourSeq 198 1381 2106 3000 81.9% chr15 - 81789869 81790408 540 browser details YourSeq 195 1382 2044 3000 90.2% chr7 - 79230216 79230918 703 browser details YourSeq 186 1399 2033 3000 89.4% chr7 + 34542459 34727773 185315 browser details YourSeq 182 1360 1627 3000 87.4% chr10 + 21666532 22020221 353690 browser details YourSeq 174 1523 2109 3000 87.0% chr15 + 55247930 55248504 575 browser details YourSeq 172 1382 1690 3000 88.4% chr9 + 98332567 98332899 333 browser details YourSeq 171 1382 1639 3000 86.9% chr11 - 115851210 115851900 691 browser details YourSeq 167 1404 1736 3000 87.8% chr5 - 131412499 131412862 364 browser details YourSeq 156 1382 1612 3000 84.5% chr4 - 136125866 136126088 223 browser details YourSeq 155 1388 1627 3000 84.8% chr1 + 164925321 164925556 236 browser details YourSeq 150 1979 2179 3000 88.9% chr17 - 35677078 35677272 195 browser details YourSeq 149 1382 1607 3000 83.3% chr7 - 135348883 135349103 221 browser details YourSeq 149 1388 1627 3000 85.8% chr19 + 40507678 40507916 239 browser details YourSeq 149 1396 1736 3000 90.4% chr11 + 102473466 102473872 407 browser details YourSeq 148 1388 1611 3000 83.2% chr9 - 64539354 64539573 220 browser details YourSeq 148 1390 1627 3000 82.6% chr10 - 77214021 77214253 233 browser details YourSeq 146 1382 1701 3000 88.4% chr1 - 37629156 37629489 334 browser details YourSeq 146 1382 1626 3000 85.5% chr10 + 24222066 24222301 236 browser details YourSeq 145 1403 1608 3000 88.1% chr10 - 76933262 77203619 270358

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% chr2 - 70990234 70993233 3000 browser details YourSeq 165 686 2239 3000 91.8% chr10 + 77031159 77222535 191377 browser details YourSeq 155 730 2294 3000 91.0% chr16 - 35899616 36122826 223211 browser details YourSeq 146 2132 2312 3000 91.0% chr17 + 65431198 65431384 187 browser details YourSeq 142 2130 2303 3000 89.0% chr13 - 45601138 45601309 172 browser details YourSeq 140 2130 2306 3000 87.0% chr16 - 21639548 21639716 169 browser details YourSeq 138 2130 2304 3000 87.6% chr14 - 124501897 124502066 170 browser details YourSeq 134 2130 2304 3000 85.8% chr11 - 22624571 22624739 169 browser details YourSeq 132 2130 2303 3000 86.8% chr4 - 11313839 11314011 173 browser details YourSeq 132 2132 2304 3000 87.8% chr15 + 79341207 79341374 168 browser details YourSeq 132 2131 2302 3000 88.9% chr14 + 80542392 80542568 177 browser details YourSeq 131 2133 2307 3000 91.7% chr14 - 9625952 9626148 197 browser details YourSeq 130 2130 2290 3000 90.7% chr5 - 132569450 132569611 162 browser details YourSeq 130 2132 2304 3000 85.7% chr10 - 109624303 109624470 168 browser details YourSeq 128 2123 2279 3000 91.8% chr4 - 126993192 126993829 638 browser details YourSeq 127 2131 2303 3000 84.1% chr5 - 141288622 141288785 164 browser details YourSeq 127 2133 2279 3000 91.8% chr16 + 30551788 30551933 146 browser details YourSeq 126 2137 2294 3000 89.7% chr4 - 3249753 3249906 154 browser details YourSeq 124 2133 2303 3000 86.4% chr4 - 4413571 4413738 168 browser details YourSeq 124 2130 2279 3000 92.0% chr2 + 155661992 155662143 152

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

Page 4 of 8 https://www.alphaknockout.com

Gene and protein information: Mettl8 methyltransferase like 8 [ Mus musculus (house mouse) ] Gene ID: 228019, updated on 8-Oct-2019

Gene summary

Official Symbol Mettl8 provided by MGI Official Full Name methyltransferase like 8 provided by MGI Primary source MGI:MGI:2385142 See related Ensembl:ENSMUSG00000041975 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Tip; BC004636 Summary This locus encodes a member of the methyltransferase family, and is involved in chromatin remodeling. Transcripts from Expression this locus can be induced or inhibited by cell stretch and affect cell differentiation in the myogenic or adipogenic pathways. Multiple transcript variants encoding different isoforms have been found for this gene. Additional splice variants have been described in the literature but they meet nonsense-mediated decay (NMD) criteria and are likely to be degraded as soon as they are transcribed. [provided by RefSeq, Jul 2008] Orthologs Ubiquitous expression in thymus adult (RPKM 2.3), CNS E11.5 (RPKM 2.1) and 28 other tissues See more human all

Genomic context

Location: 2; 2 C2 See Mettl8 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (70964561..71056388, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (70802618..70893663, complement)

Chromosome 2 - NC_000068.7

Page 5 of 8 https://www.alphaknockout.com

Transcript information: This gene has 11 transcripts

Gene: Mettl8 ENSMUSG00000041975

Description methyltransferase like 8 [Source:MGI Symbol;Acc:MGI:2385142] Gene Synonyms TIP Location Chromosome 2: 70,964,561-71,055,583 reverse strand. GRCm38:CM000995.2 About this gene This gene has 11 transcripts (splice variants), 188 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mettl8- ENSMUST00000112186.8 2347 388aa ENSMUSP00000107804.2 Protein coding CCDS50600 A2AUU0 TSL:2 204 GENCODE basic APPRIS P2

Mettl8- ENSMUST00000100037.8 2254 341aa ENSMUSP00000097615.2 Protein coding - B0R0U1 TSL:1 202 GENCODE basic APPRIS ALT2

Mettl8- ENSMUST00000090849.5 1842 163aa ENSMUSP00000088359.5 Protein coding - Q8CA70 TSL:1 201 GENCODE basic

Mettl8- ENSMUST00000121586.7 1151 281aa ENSMUSP00000113642.1 Protein coding - A2AUU0 TSL:5 205 GENCODE basic

Mettl8- ENSMUST00000140293.1 592 110aa ENSMUSP00000118026.1 Protein coding - F6W103 CDS 5' 209 incomplete TSL:3

Mettl8- ENSMUST00000148876.7 2473 304aa ENSMUSP00000115855.1 Nonsense mediated - A2AUU0 TSL:1 210 decay

Mettl8- ENSMUST00000112179.8 2297 206aa ENSMUSP00000107800.2 Nonsense mediated - A2AUU0 TSL:2 203 decay

Mettl8- ENSMUST00000149181.7 743 148aa ENSMUSP00000119863.1 Nonsense mediated - F6Q7P7 CDS 5' 211 decay incomplete TSL:5

Mettl8- ENSMUST00000124208.1 1597 No - Retained intron - - TSL:1 206 protein

Mettl8- ENSMUST00000124781.1 833 No - Retained intron - - TSL:5 208 protein

Mettl8- ENSMUST00000124273.1 390 No - Retained intron - - TSL:5 207 protein

Page 6 of 8 https://www.alphaknockout.com

111.02 kb Forward strand

70.96Mb 70.98Mb 71.00Mb 71.02Mb 71.04Mb 71.06Mb Genes Dcaf17-204 >nonsense mediated decay (Comprehensive set...

Dcaf17-208 >nonsense mediated decay

Dcaf17-205 >protein coding

Dcaf17-209 >protein coding

Dcaf17-207 >protein coding

Dcaf17-201 >protein coding

Dcaf17-203 >protein coding

Dcaf17-202 >protein coding

Contigs AL929228.5 > Genes (Comprehensive set... < Mettl8-204protein coding

< Mettl8-210nonsense mediated decay

< Mettl8-203nonsense mediated decay

< Mettl8-202protein coding

< Mettl8-205protein coding

< Mettl8-209protein coding< Mettl8-206retained intron

< Mettl8-211nonsense mediated decay < Mettl8-207retained intron

< Mettl8-208retained intron

< Mettl8-201protein coding

Regulatory Build

70.96Mb 70.98Mb 71.00Mb 71.02Mb 71.04Mb 71.06Mb Reverse strand 111.02 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000112186

< Mettl8-204protein coding

Reverse strand 91.02 kb

ENSMUSP00000107... MobiDB lite Low complexity (Seg) Superfamily S-adenosyl-L-methionine-dependent methyltransferase Pfam Methyltransferase domain 25 PIRSF Methyltransferase-like PANTHER Methyltransferase-like

PTHR22809:SF3 Gene3D 3.40.50.150 CDD cd02440

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

Variant Legend stop gained missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 388

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

Page 8 of 8