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Mouse Myt1l Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Myt1l conditional 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 14 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Myt1l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-243D15 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 14 starts from about 51.14% of the coding region. The knockout of Exon 14 will result in frameshift of the gene. The size of intron 13 for 5'-loxP site insertion: 4247 bp, and the size of intron 14 for 3'-loxP site insertion: 6635 bp. The size of effective cKO region: ~715 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 14 25 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Myt1l 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(7215bp) | A(26.92% 1942) | C(21.91% 1581) | T(29.79% 2149) | G(21.39% 1543)

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% chr12 + 29839146 29842145 3000 browser details YourSeq 690 1420 2327 3000 89.8% chr5 - 22168440 22381345 212906 browser details YourSeq 683 1420 2327 3000 89.1% chr5 + 4048034 4048972 939 browser details YourSeq 676 1420 2325 3000 89.8% chr3 + 154714251 154715187 937 browser details YourSeq 674 1416 2327 3000 89.7% chr18 - 10507187 10508325 1139 browser details YourSeq 674 1396 2327 3000 88.5% chr15 - 10652033 10652976 944 browser details YourSeq 673 1420 2327 3000 88.3% chr18 + 79880197 79881126 930 browser details YourSeq 672 1420 2327 3000 88.5% chr5 - 44407629 44733208 325580 browser details YourSeq 670 1420 2327 3000 88.9% chr14 - 69079191 69080127 937 browser details YourSeq 670 1424 2327 3000 89.5% chr1 + 159436354 159437286 933 browser details YourSeq 667 1420 2327 3000 89.8% chr14 - 70367088 70368021 934 browser details YourSeq 666 1420 2327 3000 89.1% chr7 - 96964102 96965039 938 browser details YourSeq 666 1394 2327 3000 89.4% chr11 + 65314760 65315708 949 browser details YourSeq 664 1420 2327 3000 88.7% chr17 - 49749094 49750030 937 browser details YourSeq 664 1420 2327 3000 89.3% chrX + 151692714 151693651 938 browser details YourSeq 662 1420 2307 3000 89.2% chr13 + 91925161 91926077 917 browser details YourSeq 661 1420 2327 3000 88.9% chr15 + 91743736 91744675 940 browser details YourSeq 659 1421 2329 3000 88.1% chr2 + 19100150 19101086 937 browser details YourSeq 657 1421 2327 3000 88.4% chr9 - 35602896 35603831 936 browser details YourSeq 657 1420 2327 3000 88.0% chr18 - 79346196 79347134 939

Note: The 3000 bp section upstream of Exon 14 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% chr12 + 29842861 29845860 3000 browser details YourSeq 24 197 220 3000 100.0% chrX - 162172739 162172762 24 browser details YourSeq 24 1143 1169 3000 96.2% chr6 - 12795833 12795861 29 browser details YourSeq 22 2675 2696 3000 100.0% chr4 - 105787250 105787271 22 browser details YourSeq 22 2845 2869 3000 95.9% chr18 - 58657571 58657596 26 browser details YourSeq 21 2837 2857 3000 100.0% chr1_GL456221_random - 204871 204891 21 browser details YourSeq 21 1500 1521 3000 100.0% chr1 - 14811984 14812006 23 browser details YourSeq 20 529 552 3000 91.7% chr10 - 108465555 108465578 24

Note: The 3000 bp section downstream of Exon 14 is BLAT searched against the genome. No significant similarity is found.

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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

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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

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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

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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.

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