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Mouse Tln2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tln2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tln2 gene (NCBI Reference Sequence: NM_001081242 ; Ensembl: ENSMUSG00000052698 ) is located on Mouse chromosome 9. 58 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 58 (Transcript: ENSMUST00000040025). Exon 8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tln2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-84P10 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 abnormal muscle morphology.

Exon 8 starts from about 8.67% of the coding region. The knockout of Exon 8 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 1885 bp, and the size of intron 8 for 3'-loxP site insertion: 9686 bp. The size of effective cKO region: ~628 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 7 8 58 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tln2 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(7128bp) | A(26.02% 1855) | C(21.2% 1511) | T(29.85% 2128) | G(22.92% 1634)

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% chr9 - 67386935 67389934 3000 browser details YourSeq 328 10 695 3000 83.7% chr12 - 69008121 69008921 801 browser details YourSeq 327 10 566 3000 86.1% chr2 + 129423895 129424491 597 browser details YourSeq 304 85 645 3000 87.1% chr13 - 22403315 23017871 614557 browser details YourSeq 304 10 647 3000 88.7% chr18 + 46953153 46954352 1200 browser details YourSeq 302 94 660 3000 86.8% chr16 + 55889443 55890300 858 browser details YourSeq 284 82 646 3000 86.0% chr1 - 44518756 44519512 757 browser details YourSeq 279 77 643 3000 85.5% chr1 - 123266938 123267776 839 browser details YourSeq 277 94 800 3000 87.9% chrX + 95041750 95042519 770 browser details YourSeq 276 10 694 3000 86.2% chr7 + 140831574 140832328 755 browser details YourSeq 269 92 597 3000 84.4% chr6 - 73243186 73243696 511 browser details YourSeq 269 82 646 3000 87.8% chr19 - 29822314 29823121 808 browser details YourSeq 267 14 693 3000 81.6% chr9 - 9564140 9564781 642 browser details YourSeq 263 10 644 3000 87.6% chr14 - 64780757 64970108 189352 browser details YourSeq 263 228 893 3000 86.8% chr11 + 3168066 3168732 667 browser details YourSeq 262 10 530 3000 85.7% chr7 + 127080726 127081243 518 browser details YourSeq 262 10 642 3000 85.2% chr5 + 49860360 49861080 721 browser details YourSeq 258 10 698 3000 87.1% chr3 + 63635341 63636414 1074 browser details YourSeq 258 10 644 3000 80.9% chr1 + 80544902 80545756 855 browser details YourSeq 256 11 695 3000 82.3% chr9 - 52980859 52981457 599

Note: The 3000 bp section upstream of Exon 8 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% chr9 - 67383307 67386306 3000 browser details YourSeq 74 2293 2370 3000 98.8% chr6 + 86446714 86446883 170 browser details YourSeq 74 2294 2378 3000 94.2% chr12 + 72218687 72218789 103 browser details YourSeq 73 2294 2366 3000 100.0% chr3 - 42021009 42021081 73 browser details YourSeq 73 2294 2370 3000 98.8% chr11 - 21088181 21088429 249 browser details YourSeq 73 2294 2372 3000 97.5% chr10 - 63409964 63410177 214 browser details YourSeq 73 2294 2370 3000 97.5% chr3 + 121161279 121161355 77 browser details YourSeq 73 2294 2375 3000 97.6% chr1 + 178568500 178568647 148 browser details YourSeq 72 2294 2370 3000 98.8% chr14 + 7030413 7030654 242 browser details YourSeq 71 2294 2370 3000 97.4% chr8 - 117744411 117744491 81 browser details YourSeq 71 2292 2368 3000 97.5% chr18 - 83251478 83251758 281 browser details YourSeq 71 2294 2368 3000 98.7% chr2 + 35973945 35974035 91 browser details YourSeq 71 2293 2370 3000 97.5% chr15 + 38348418 38348679 262 browser details YourSeq 70 2294 2368 3000 98.7% chr5 - 89654956 89655108 153 browser details YourSeq 70 2293 2368 3000 98.7% chr2 - 5491129 5491240 112 browser details YourSeq 70 2294 2370 3000 97.4% chrX + 104016268 104016358 91 browser details YourSeq 70 2294 2370 3000 97.5% chr6 + 102778657 102778743 87 browser details YourSeq 70 2294 2370 3000 96.1% chr15 + 41151190 41151276 87 browser details YourSeq 70 2294 2366 3000 98.7% chr14 + 72986977 72987061 85 browser details YourSeq 70 2294 2370 3000 98.8% chr14 + 5884740 5884910 171

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tln2 talin 2 [ Mus musculus (house mouse) ] Gene ID: 70549, updated on 12-Aug-2019

Gene summary

Official Symbol Tln2 provided by MGI Official Full Name talin 2 provided by MGI Primary source MGI:MGI:1917799 See related Ensembl:ENSMUSG00000052698 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 AI507121; AI787438; AL118320; mKIAA0320; 5730421P04Rik Expression Broad expression in kidney adult (RPKM 19.0), testis adult (RPKM 15.4) and 20 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 C See Tln2 in Genome Data Viewer

Exon count: 63

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (67217085..67634045, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (67064892..67407510, complement)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 9 transcripts

Gene: Tln2 ENSMUSG00000052698

Description talin 2 [Source:MGI Symbol;Acc:MGI:1917799] Location Chromosome 9: 67,217,087-67,559,703 reverse strand. GRCm38:CM001002.2 About this gene This gene has 9 transcripts (splice variants), 283 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tln2-202 ENSMUST00000040025.13 12132 2542aa ENSMUSP00000039633.7 Protein coding CCDS52849 E9PUM4 TSL:5 GENCODE basic APPRIS P2

Tln2-201 ENSMUST00000039662.8 8052 2542aa ENSMUSP00000035272.8 Protein coding CCDS52849 E9PUM4 TSL:1 GENCODE basic APPRIS P2

Tln2-207 ENSMUST00000215784.1 8330 2544aa ENSMUSP00000148901.1 Protein coding - A0A1L1SQ51 TSL:5 GENCODE basic APPRIS ALT1

Tln2-209 ENSMUST00000217550.1 5152 1471aa ENSMUSP00000149474.1 Protein coding - A0A1L1SRI1 CDS 5' incomplete TSL:1

Tln2-205 ENSMUST00000215267.1 4999 1452aa ENSMUSP00000149284.1 Protein coding - Q8CDM9 TSL:1 GENCODE basic

Tln2-204 ENSMUST00000214859.1 3251 923aa ENSMUSP00000149137.1 Protein coding - A0A1L1SQP9 CDS 5' incomplete TSL:1

Tln2-203 ENSMUST00000213584.1 582 137aa ENSMUSP00000151111.1 Protein coding - A0A1L1SVA8 CDS 3' incomplete TSL:1

Tln2-208 ENSMUST00000216799.1 3365 No protein - Retained intron - - TSL:NA

Tln2-206 ENSMUST00000215593.1 3161 No protein - lncRNA - - TSL:1

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362.62 kb Forward strand

67.3Mb 67.4Mb 67.5Mb Genes Gm19299-201 >lncRNA Gm47047-201 >lncRNA (Comprehensive set...

Contigs < AC107740.17 AC173343.1 > Genes (Comprehensive set... < Tln2-202protein coding

< Tln2-209protein coding < Tln2-203protein coding

< Tln2-207protein coding

< Tln2-204protein coding

< Tln2-205protein coding

< Tln2-201protein coding

< Tln2-208retained intron

< Mir190a-201miRNA

< Tln2-206lncRNA

Regulatory Build

67.3Mb 67.4Mb 67.5Mb Reverse strand 362.62 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000040025

< Tln2-202protein coding

Reverse strand 342.62 kb

ENSMUSP00000039... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 I/LWEQ domain superfamily

Ubiquitin-like domain superfamily Alpha-catenin/vinculin-like superfamily

Talin, central domain superfamily

FERM superfamily, second domain SMART SM01244 I/LWEQ domain

Band 4.1 domain Pfam FERM, N-terminal I/LWEQ domain

Talin, central Vinculin-binding site-containing domain

Talin, N-terminal F0 domain

FERM central domain PROSITE profiles FERM domain I/LWEQ domain

PROSITE patterns FERM conserved site PANTHER PTHR19981:SF34

PTHR19981 Gene3D 3.10.20.90 PH-like domain superfamily 1.20.1410.10

1.20.120.230

FERM/acyl-CoA-binding protein superfamily

1.20.1440.10

1.20.1420.10 CDD cd17172 cd10569 Talin-1/2, rod-segment

FERM central domain

cd17174

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 400 800 1200 1600 2000 2542

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

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