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Mouse Krt86 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Krt86 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Krt86 gene (NCBI Reference Sequence: NM_010667 ; Ensembl: ENSMUSG00000067614 ) is located on Mouse chromosome 15. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000088049). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Krt86 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-79F3 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 2 starts from about 25.38% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 620 bp, and the size of intron 2 for 3'-loxP site insertion: 656 bp. The size of effective cKO region: ~709 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 2 3 4 5 6 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Krt86 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(7209bp) | A(23.96% 1727) | C(24.44% 1762) | T(24.87% 1793) | G(26.73% 1927)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr15 + 101471291 101474290 3000 browser details YourSeq 656 2293 3000 3000 97.7% chr15 - 101462985 101491423 28439 browser details YourSeq 348 2283 2648 3000 97.6% chr15 - 101463310 101463675 366 browser details YourSeq 330 2291 2654 3000 95.4% chr15 - 101438362 101438725 364 browser details YourSeq 112 2508 2640 3000 91.5% chr15 + 101503768 101503898 131 browser details YourSeq 72 2460 2631 3000 72.7% chr15 - 101712272 101712419 148 browser details YourSeq 72 2514 2631 3000 80.6% chr15 - 101693740 101693857 118 browser details YourSeq 31 2636 2678 3000 88.3% chr11 - 59283126 59283166 41 browser details YourSeq 23 920 945 3000 96.2% chr11 + 8808070 8808096 27

Note: The 3000 bp section upstream of Exon 2 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% chr15 + 101475000 101477999 3000 browser details YourSeq 1273 407 2476 3000 95.2% chr15 - 101460121 101461821 1701 browser details YourSeq 1177 1199 2482 3000 95.9% chr15 - 101486964 101488248 1285 browser details YourSeq 998 1200 2476 3000 95.6% chr15 - 101433099 101434678 1580 browser details YourSeq 203 1485 2476 3000 88.7% chr15 - 101542687 101678152 135466 browser details YourSeq 181 1485 2467 3000 82.1% chr15 - 101527711 101692331 164621 browser details YourSeq 112 2324 2468 3000 89.0% chr15 - 101708881 101756730 47850 browser details YourSeq 106 2323 2479 3000 84.1% chr15 - 101555254 101691435 136182 browser details YourSeq 103 2313 2479 3000 80.9% chr15 - 101997847 101998013 167 browser details YourSeq 98 2313 2468 3000 81.5% chr7 + 23358169 23358324 156 browser details YourSeq 89 2260 2468 3000 73.2% chr15 + 101423352 101423485 134 browser details YourSeq 87 2336 2476 3000 80.9% chr15 + 101452459 101452599 141 browser details YourSeq 84 2349 2468 3000 85.0% chr15 - 101887303 101887422 120 browser details YourSeq 76 2349 2468 3000 81.7% chr15 - 101919541 101919660 120 browser details YourSeq 66 2323 2418 3000 84.4% chr15 - 101677161 101677256 96 browser details YourSeq 65 2345 2447 3000 81.6% chr15 + 101609795 101609897 103 browser details YourSeq 50 2262 2373 3000 90.4% chr3 + 75392237 75392352 116 browser details YourSeq 49 2388 2476 3000 77.6% chr3 - 88484924 88485012 89 browser details YourSeq 37 2335 2375 3000 95.2% chr15 - 101351094 101351134 41 browser details YourSeq 29 2260 2374 3000 54.9% chr15 - 101948520 101948559 40

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Krt86 keratin 86 [ Mus musculus (house mouse) ] Gene ID: 16679, updated on 12-Aug-2019

Gene summary

Official Symbol Krt86 provided by MGI Official Full Name keratin 86 provided by MGI Primary source MGI:MGI:109362 See related Ensembl:ENSMUSG00000067614 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Khb4; MHb4; Krt2-10; Krt2-11; AI325212; 5430421N21Rik Expression Low expression observed in reference dataset See more

Genomic context

Location: 15 F2; 15 56.9 cM See Krt86 in Genome Data Viewer Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (101473478..101479983)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (101303909..101310414)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 1 transcript

Gene: Krt86 ENSMUSG00000067614

Description keratin 86 [Source:MGI Symbol;Acc:MGI:109362] Gene Synonyms Khb4, Krt2-10, Krt2-11, MHb4 Location Chromosome 15: 101,473,478-101,479,986 forward strand. GRCm38:CM001008.2 About this gene This gene has 1 transcript (splice variant), 139 orthologues, 68 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Krt86-201 ENSMUST00000088049.4 1963 486aa ENSMUSP00000085365.3 Protein coding CCDS37218 B2RTB6 P97861 TSL:1 GENCODE basic APPRIS P1

26.51 kb Forward strand

101.465Mb 101.470Mb 101.475Mb 101.480Mb 101.485Mb Genes (Comprehensive set... Krt86-201 >protein coding Gm49501-201 >lncRNA

Contigs AC103674.10 > Genes < Krt81-201protein coding < Krt83-201protein coding (Comprehensive set...

< Krt81-202retained intron < Krt83-202retained intron

Regulatory Build

101.465Mb 101.470Mb 101.475Mb 101.480Mb 101.485Mb Reverse strand 26.51 kb

Regulation Legend

CTCF Enhancer Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000088049

6.51 kb Forward strand

Krt86-201 >protein coding

ENSMUSP00000085... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Growth factor receptor cysteine-rich domain superfamily

SSF64593 SMART Intermediate filament, rod domain

Prints Keratin, type II Pfam Keratin type II head

Intermediate filament, rod domain PROSITE profiles Intermediate filament, rod domain

PROSITE patterns Intermediate filament protein, conserved site

PANTHER PTHR45616

PTHR45616:SF27 Gene3D Intermediate filament, rod domain, coil 1B 1.20.5.170

1.20.5.500

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 486

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

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