https://www.alphaknockout.com

Mouse Ttll3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ttll3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ttll3 gene (NCBI Reference Sequence: NM_133923 ; Ensembl: ENSMUSG00000030276 ) is located on Mouse chromosome 6. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000032414). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ttll3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-339P1 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 a reduced number of primary cilia in colon epithelia accompanied by an increased rate of cell division which is compensated by faster tissue turnover in the colon. Mice exhibit increased incidence of colon tumors by chemical induction.

Exon 5 starts from about 27.33% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2834 bp, and the size of intron 6 for 3'-loxP site insertion: 782 bp. The size of effective cKO region: ~1757 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 5' gRNA region gRNA region 3'

1 5 6 7 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ttll3 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. 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(7955bp) | A(24.46% 1946) | C(25.36% 2017) | T(25.09% 1996) | G(25.09% 1996)

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% chr6 + 113394681 113397680 3000 browser details YourSeq 278 981 2100 3000 95.5% chr1 + 59820296 60175985 355690 browser details YourSeq 237 1392 2493 3000 94.8% chr2 + 126952304 127382875 430572 browser details YourSeq 172 1956 2491 3000 87.2% chr17 - 24307864 24308228 365 browser details YourSeq 157 1838 2082 3000 87.8% chr2 - 127185564 127185805 242 browser details YourSeq 156 1910 2101 3000 92.1% chr16 - 32318738 32318932 195 browser details YourSeq 144 1939 2102 3000 93.2% chr15 - 36411313 36411475 163 browser details YourSeq 142 1951 2102 3000 96.8% chr1 + 54039516 54039667 152 browser details YourSeq 141 1929 2101 3000 94.4% chr14 + 47671051 47671223 173 browser details YourSeq 138 1951 2103 3000 95.4% chr13 + 4365303 4365475 173 browser details YourSeq 137 1952 2101 3000 96.0% chr4 - 50769899 50770048 150 browser details YourSeq 137 1889 2088 3000 86.5% chr18 + 75729763 75729917 155 browser details YourSeq 136 1959 2100 3000 97.9% chr13 - 14205181 14205322 142 browser details YourSeq 136 1951 2100 3000 94.6% chr2 + 118954452 118954600 149 browser details YourSeq 136 1951 2102 3000 95.4% chr11 + 107612018 107612185 168 browser details YourSeq 135 1951 2105 3000 94.2% chr8 - 37589472 37589630 159 browser details YourSeq 135 1939 2105 3000 93.6% chr11 + 112221090 112221261 172 browser details YourSeq 133 1951 2099 3000 94.7% chr4 + 117781267 117781415 149 browser details YourSeq 132 1951 2103 3000 94.0% chr2 + 115806394 115806549 156 browser details YourSeq 132 1960 2103 3000 95.9% chr19 + 37186371 37186514 144

Note: The 3000 bp section upstream of Exon 5 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 + 113399136 113402135 3000 browser details YourSeq 158 2576 2999 3000 87.3% chr5 - 76988837 76989040 204 browser details YourSeq 156 2835 2997 3000 98.2% chr6 - 142965589 142965752 164 browser details YourSeq 156 2831 2999 3000 96.5% chr7 + 89202688 89202857 170 browser details YourSeq 155 2833 3000 3000 96.5% chr12 - 80493062 80493230 169 browser details YourSeq 155 2832 3000 3000 95.9% chr11 + 22523810 22523978 169 browser details YourSeq 154 2832 2999 3000 96.5% chr11 - 115530654 115530824 171 browser details YourSeq 154 2832 2998 3000 96.4% chr3 + 88293075 88293253 179 browser details YourSeq 154 2832 3000 3000 95.9% chr3 + 18362550 18362719 170 browser details YourSeq 154 2827 2997 3000 96.5% chr19 + 43685311 43685491 181 browser details YourSeq 153 2832 2999 3000 95.9% chr4 - 84531589 84531758 170 browser details YourSeq 153 2832 2999 3000 95.9% chr8 + 55741819 55741989 171 browser details YourSeq 153 2832 2999 3000 95.9% chr5 + 29779714 29779882 169 browser details YourSeq 153 2832 2998 3000 95.9% chr14 + 7761620 7761786 167 browser details YourSeq 153 2832 2997 3000 96.4% chr11 + 54797459 54797625 167 browser details YourSeq 153 2827 2998 3000 95.3% chr1 + 60387423 60387598 176 browser details YourSeq 152 2832 2998 3000 95.9% chr7 - 27318431 27318598 168 browser details YourSeq 152 2827 3000 3000 94.3% chr10 - 22147967 22148205 239 browser details YourSeq 152 2832 2998 3000 95.9% chrX + 41987248 41987426 179 browser details YourSeq 152 2835 2999 3000 96.4% chr19 + 4358997 4359162 166

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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

Gene and protein information: Ttll3 tubulin tyrosine ligase-like family, member 3 [ Mus musculus (house mouse) ] Gene ID: 101100, updated on 12-Aug-2019

Gene summary

Official Symbol Ttll3 provided by MGI Official Full Name tubulin tyrosine ligase-like family, member 3 provided by MGI Primary source MGI:MGI:2141418 See related Ensembl:ENSMUSG00000030276 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 AI450050; 4833441J24Rik Expression Ubiquitous expression in testis adult (RPKM 32.1), thymus adult (RPKM 12.4) and 28 other tissues See more

Genomic context

Location: 6; 6 E3 See Ttll3 in Genome Data Viewer Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (113389260..113414592)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (113339254..113364577)

Chromosome 6 - NC_000072.6

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

Transcript information: This gene has 9 transcripts

Gene: Ttll3 ENSMUSG00000030276

Description tubulin tyrosine ligase-like family, member 3 [Source:MGI Symbol;Acc:MGI:2141418] Gene Synonyms 4833441J24Rik Location Chromosome 6: 113,389,260-113,414,587 forward strand. GRCm38:CM000999.2 About this gene This gene has 9 transcripts (splice variants), 230 orthologues, 13 paralogues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ttll3- ENSMUST00000032414.10 3168 927aa ENSMUSP00000032414.4 Protein coding CCDS39593 A4Q9E5 TSL:1 201 GENCODE basic APPRIS P2

Ttll3- ENSMUST00000204026.2 1472 287aa ENSMUSP00000145049.1 Protein coding CCDS85118 A4Q9E5 TSL:1 206 GENCODE basic

Ttll3- ENSMUST00000038889.11 3114 928aa ENSMUSP00000037870.5 Protein coding - H7BX03 TSL:5 202 GENCODE basic APPRIS ALT2

Ttll3- ENSMUST00000203524.2 2745 721aa ENSMUSP00000145329.1 Protein coding - F8VQA1 CDS 5' 203 incomplete TSL:5

Ttll3- ENSMUST00000205017.2 1886 68aa ENSMUSP00000145044.1 Nonsense mediated - F6T422 CDS 5' 209 decay incomplete TSL:1

Ttll3- ENSMUST00000203925.1 2517 No - Retained intron - - TSL:1 205 protein

Ttll3- ENSMUST00000203880.1 827 No - lncRNA - - TSL:3 204 protein

Ttll3- ENSMUST00000204683.1 782 No - lncRNA - - TSL:5 208 protein

Ttll3- ENSMUST00000204255.1 396 No - lncRNA - - TSL:3 207 protein

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

45.33 kb Forward strand

113.38Mb 113.39Mb 113.40Mb 113.41Mb 113.42Mb Genes (Comprehensive set... Arpc4-201 >protein coding Ttll3-208 >lncRNA Ttll3-207 >lncRNA Gm44280-201 >TEC

Arpc4-203 >protein coding Ttll3-201 >protein coding Gm24387-201 >miRNA

Arpc4-202 >protein coding Ttll3-206 >protein coding Ttll3-204 >lncRNA

Arpc4-204 >protein coding Ttll3-202 >protein coding

Ttll3-203 >protein coding

Ttll3-209 >nonsense mediated decay

Ttll3-205 >retained intron

Contigs AC155287.6 > AC153910.6 > Genes < Rpusd3-202protein coding (Comprehensive set...

< Rpusd3-201protein coding

< Rpusd3-210retained intron

< Rpusd3-204protein coding

< Rpusd3-207protein coding

< Rpusd3-203retained intron

< Rpusd3-209protein coding

< Rpusd3-206retained intron

< Rpusd3-205protein coding

< Rpusd3-208retained intron

Regulatory Build

113.38Mb 113.39Mb 113.40Mb 113.41Mb 113.42Mb Reverse strand 45.33 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

RNA gene processed transcript

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

Transcript: ENSMUST00000032414

22.13 kb Forward strand

Ttll3-201 >protein coding

ENSMUSP00000032... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF56059 Pfam Tubulin-tyrosine ligase/Tubulin polyglutamylase PROSITE profiles Tubulin-tyrosine ligase/Tubulin polyglutamylase PANTHER PTHR45870

Tubulin monoglycylase TTLL3 Gene3D 3.30.470.20

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

Variant Legend stop gained missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 927

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

Page 8 of 8