https://www.alphaknockout.com Mouse Myt1l Knockout Project (CRISPR/Cas9) Objective: To create a Myt1l knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Myt1l gene (NCBI Reference Sequence: NM_001093775 ; Ensembl: ENSMUSG00000061911 ) is located on Mouse chromosome 12. 25 exons are identified, with the ATG start codon in exon 6 and the TGA stop codon in exon 25 (Transcript: ENSMUST00000049784). Exon 9 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: Exon 9 starts from about 4.3% of the coding region. Exon 9 covers 10.17% of the coding region. The size of effective KO region: ~362 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 9 25 Legends Exon of mouse Myt1l Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 9 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 9 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(27.6% 552) | C(23.85% 477) | T(29.3% 586) | G(19.25% 385) Note: The 2000 bp section upstream of Exon 9 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(29.8% 596) | C(18.9% 378) | T(30.35% 607) | G(20.95% 419) Note: The 2000 bp section downstream of Exon 9 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 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr12 + 29809374 29811373 2000 browser details YourSeq 23 1966 1988 2000 100.0% chr4 - 103475909 103475931 23 browser details YourSeq 22 289 310 2000 100.0% chr16 - 3796270 3796291 22 browser details YourSeq 22 796 819 2000 87.0% chr1 + 106132106 106132128 23 browser details YourSeq 21 630 650 2000 100.0% chr7 + 54998016 54998036 21 Note: The 2000 bp section upstream of Exon 9 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% chr12 + 29811736 29813735 2000 browser details YourSeq 35 698 752 2000 94.9% chr1 - 22652140 22652207 68 browser details YourSeq 29 314 350 2000 87.5% chr1 - 25216747 25216782 36 browser details YourSeq 22 225 246 2000 100.0% chr13 - 101466696 101466717 22 browser details YourSeq 22 640 662 2000 100.0% chr1 + 40152623 40152646 24 browser details YourSeq 21 410 430 2000 100.0% chr2 - 108914001 108914021 21 Note: The 2000 bp section downstream of Exon 9 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Myt1l myelin transcription factor 1-like [ Mus musculus (house mouse) ] Gene ID: 17933, updated on 24-Oct-2019 Gene summary Official Symbol Myt1l provided by MGI Official Full Name myelin transcription factor 1-like provided by MGI Primary source MGI:MGI:1100511 See related Ensembl:ENSMUSG00000061911 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 Nztf1; Pmng1; Png-1; mKIAA1106; 2900046C06Rik; 2900093J19Rik; C630034G21Rik Expression Biased expression in CNS E18 (RPKM 9.3), frontal lobe adult (RPKM 8.6) and 6 other tissues See more Orthologs human all Genomic context Location: 12 A2; 12 11.86 cM See Myt1l in Genome Data Viewer Exon count: 29 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (29527590..29923216) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (30213249..30608074) Chromosome 12 - NC_000078.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Myt1l ENSMUSG00000061911 Description myelin transcription factor 1-like [Source:MGI Symbol;Acc:MGI:1100511] Gene Synonyms 2900046C06Rik, 2900093J19Rik, C630034G21Rik, Nztf1, Pmng1, Png-1 Location Chromosome 12: 29,528,384-29,923,213 forward strand. GRCm38:CM001005.2 About this gene This gene has 11 transcripts (splice variants), 271 orthologues, 5 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Myt1l-202 ENSMUST00000049784.16 7202 1187aa ENSMUSP00000058264.9 Protein coding CCDS49042 P97500 TSL:1 GENCODE basic APPRIS P4 Myt1l-201 ENSMUST00000021009.9 5027 1185aa ENSMUSP00000021009.8 Protein coding CCDS49041 P97500 TSL:1 GENCODE basic APPRIS ALT2 Myt1l-205 ENSMUST00000218583.1 4522 1185aa ENSMUSP00000151588.1 Protein coding CCDS49041 P97500 TSL:1 GENCODE basic APPRIS ALT2 Myt1l-204 ENSMUST00000218198.1 2735 328aa ENSMUSP00000151919.1 Protein coding - P97500 TSL:1 GENCODE basic Myt1l-209 ENSMUST00000219744.1 796 189aa ENSMUSP00000151977.1 Protein coding - A0A1W2P887 CDS 5' incomplete TSL:3 Myt1l-206 ENSMUST00000219060.1 3179 No protein - Retained intron - - TSL:NA Myt1l-203 ENSMUST00000217961.1 1612 No protein - Retained intron - - TSL:5 Myt1l-207 ENSMUST00000219231.1 476 No protein - Retained intron - - TSL:3 Myt1l-210 ENSMUST00000219899.1 448 No protein - Retained intron - - TSL:2 Myt1l-208 ENSMUST00000219463.1 632 No protein - lncRNA - - TSL:3 Myt1l-211 ENSMUST00000220072.1 504 No protein - lncRNA - - TSL:5 Page 7 of 9 https://www.alphaknockout.com 414.83 kb Forward strand 29.6Mb 29.7Mb 29.8Mb 29.9Mb Genes (Comprehensive set... Myt1l-202 >protein coding Myt1l-205 >protein coding Myt1l-206 >retained intron Myt1l-204 >protein coding Myt1l-210 >retained intron Myt1l-211 >lncRNA Myt1l-207 >retained intron Myt1l-209 >protein coding Myt1l-201 >protein coding Myt1l-208 >lncRNA Myt1l-203 >retained intron Contigs < CT025654.11 < AC166936.1 < AC154318.3 AC154783.2 > < AC159626.2 Genes < Gm20208-202lncRNA < C630031E19Rik-201lncRNA (Comprehensive set... < Gm20208-201lncRNA Regulatory Build 29.6Mb 29.7Mb 29.8Mb 29.9Mb Reverse strand 414.83 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000049784 394.83 kb Forward strand Myt1l-202 >protein coding ENSMUSP00000058... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Zinc finger, C2H2C-type superfamily Pfam Zinc finger, C2H2C-type Myelin transcription factor 1 PROSITE profiles Zinc finger, C2H2C-type PANTHER PTHR10816 PTHR10816:SF11 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1000 1187 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.