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Mouse Kcmf1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Kcmf1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kcmf1 gene (NCBI Reference Sequence: NM_019715 ; Ensembl: ENSMUSG00000055239 ) is located on Mouse chromosome 6. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000068697). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kcmf1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-335P11 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 gene trapped allele exhibit some perinatal and postnatal lethality but mice that survive to adulthood exhibit normal lethality.

Exon 3 starts from about 16.19% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 2844 bp, and the size of intron 3 for 3'-loxP site insertion: 8289 bp. The size of effective cKO region: ~640 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 3 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Kcmf1 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. 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(7140bp) | A(27.1% 1935) | C(19.65% 1403) | T(33.29% 2377) | G(19.96% 1425)

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% chr6 - 72859164 72862163 3000 browser details YourSeq 165 2435 2619 3000 97.2% chr10 - 41471570 41794710 323141 browser details YourSeq 146 2435 2619 3000 92.6% chr13 - 61759066 62188580 429515 browser details YourSeq 137 2440 2589 3000 97.4% chr10 - 63030350 63195198 164849 browser details YourSeq 128 2435 2607 3000 93.3% chr11 + 95718225 95718528 304 browser details YourSeq 122 2435 2603 3000 92.5% chr13 - 54612939 54613196 258 browser details YourSeq 102 2435 2618 3000 81.4% chr4 + 33309617 33309734 118 browser details YourSeq 102 2435 2580 3000 91.9% chr2 + 122122404 122122792 389 browser details YourSeq 101 2435 2552 3000 94.0% chr6 + 108742111 108742232 122 browser details YourSeq 100 2435 2617 3000 83.4% chr14 + 96414375 96414500 126 browser details YourSeq 100 2435 2621 3000 84.8% chr11 + 80073001 80073105 105 browser details YourSeq 99 2435 2546 3000 91.9% chr8 + 112934925 112935034 110 browser details YourSeq 99 2370 2543 3000 92.3% chr11 + 78446355 78446809 455 browser details YourSeq 99 2428 2546 3000 95.5% chr10 + 70302480 70302602 123 browser details YourSeq 96 2435 2541 3000 95.4% chr11 - 45982125 45982232 108 browser details YourSeq 96 2428 2546 3000 87.9% chr10 - 81111701 81111811 111 browser details YourSeq 96 2435 2546 3000 90.1% chr1 - 58422261 58422361 101 browser details YourSeq 96 2435 2542 3000 94.5% chr5 + 149652162 149652269 108 browser details YourSeq 96 2435 2618 3000 84.2% chr15 + 12845546 12845646 101 browser details YourSeq 96 2435 2546 3000 90.1% chr13 + 26767826 26767926 101

Note: The 3000 bp section upstream of Exon 3 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 - 72855524 72858523 3000 browser details YourSeq 157 2722 3000 3000 86.5% chr15 - 88964772 88965084 313 browser details YourSeq 141 2720 2919 3000 86.9% chr4 - 44283754 44283961 208 browser details YourSeq 138 2724 3000 3000 87.5% chr16 - 22557008 22748907 191900 browser details YourSeq 134 2720 3000 3000 89.8% chr17 + 29915151 29915461 311 browser details YourSeq 130 2718 2882 3000 88.9% chr11 + 105098799 105098962 164 browser details YourSeq 129 2720 2886 3000 87.4% chr17 + 45181913 45182078 166 browser details YourSeq 128 2714 2894 3000 84.7% chr6 - 43579258 43579433 176 browser details YourSeq 128 2720 2888 3000 89.0% chr1 - 62664131 62664299 169 browser details YourSeq 128 2719 2894 3000 86.2% chr2 + 13960627 13960801 175 browser details YourSeq 128 2720 2894 3000 85.7% chr1 + 90438430 90438598 169 browser details YourSeq 127 2714 3000 3000 90.3% chr5 - 100314460 100314781 322 browser details YourSeq 127 2718 3000 3000 89.0% chr5 - 99216771 99217175 405 browser details YourSeq 126 2720 2894 3000 85.0% chr2 - 167204993 167205161 169 browser details YourSeq 126 2721 2894 3000 84.2% chr18 - 47043214 47043381 168 browser details YourSeq 126 2720 2894 3000 86.1% chr11 + 82689661 82689830 170 browser details YourSeq 125 2715 2894 3000 85.2% chr7 - 29738986 29739162 177 browser details YourSeq 125 2728 2893 3000 86.8% chr17 - 68010887 68011046 160 browser details YourSeq 125 2720 2885 3000 87.9% chr9 + 52516192 52516357 166 browser details YourSeq 125 2719 2889 3000 83.9% chr6 + 85845912 85846078 167

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Kcmf1 potassium channel modulatory factor 1 [ Mus musculus (house mouse) ] Gene ID: 74287, updated on 12-Aug-2019

Gene summary

Official Symbol Kcmf1 provided by MGI Official Full Name potassium channel modulatory factor 1 provided by MGI Primary source MGI:MGI:1921537 See related Ensembl:ENSMUSG00000055239 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 Pmcf; Debt91; 1700094M07Rik Expression Ubiquitous expression in testis adult (RPKM 46.7), ovary adult (RPKM 17.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 6 C1; 6 32.3 cM See Kcmf1 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (72841114..72899979, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (72791108..72849973, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 6 transcripts

Gene: Kcmf1 ENSMUSG00000055239

Description potassium channel modulatory factor 1 [Source:MGI Symbol;Acc:MGI:1921537] Gene Synonyms 1700094M07Rik, Pmcf, clone DEBT-91 Location Chromosome 6: 72,841,114-72,899,979 reverse strand. GRCm38:CM000999.2 About this gene This gene has 6 transcripts (splice variants), 254 orthologues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kcmf1-201 ENSMUST00000068697.10 3251 381aa ENSMUSP00000064410.8 Protein coding CCDS39518 Q80UY2 TSL:1 GENCODE basic APPRIS P1

Kcmf1-204 ENSMUST00000204598.2 1743 330aa ENSMUSP00000144910.1 Protein coding CCDS85066 A0A0N4SV15 TSL:1 GENCODE basic

Kcmf1-205 ENSMUST00000204708.1 549 47aa ENSMUSP00000144907.1 Protein coding - A0A0N4SV12 CDS 3' incomplete TSL:3

Kcmf1-206 ENSMUST00000206378.1 544 110aa ENSMUSP00000145559.1 Protein coding - A0A0U1RNG8 TSL:5 GENCODE basic

Kcmf1-202 ENSMUST00000203004.1 3044 No protein - Retained intron - - TSL:NA

Kcmf1-203 ENSMUST00000203431.1 393 No protein - lncRNA - - TSL:3

78.87 kb Forward strand 72.84Mb 72.86Mb 72.88Mb 72.90Mb Contigs < AC153613.9 Genes (Comprehensive set... < Kcmf1-201protein coding

< Kcmf1-204protein coding

< Kcmf1-206protein coding

< Kcmf1-202retained intron < D530018E20Rik-201TEC

< Kcmf1-203lncRNA

< Kcmf1-205protein coding

Regulatory Build

72.84Mb 72.86Mb 72.88Mb 72.90Mb Reverse strand 78.87 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

RNA gene processed transcript

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Transcript: ENSMUST00000068697

< Kcmf1-201protein coding

Reverse strand 58.87 kb

ENSMUSP00000064... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57850 SMART Zinc finger, ZZ-type Zinc finger C2H2-type

Pfam Zinc finger, ZZ-type Drought induced 19 protein type, zinc-binding domain

PROSITE profiles Zinc finger, ZZ-type Zinc finger C2H2-type

PROSITE patterns Zinc finger, ZZ-type

PANTHER E3 ubiquitin-protein ligase KCMF1

PTHR12268 Gene3D 3.30.60.90 CDD cd02338

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 381

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

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