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Mouse Farp1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Farp1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Farp1 gene (NCBI Reference Sequence: NM_134082 ; Ensembl: ENSMUSG00000025555 ) is located on Mouse chromosome 14. 27 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 27 (Transcript: ENSMUST00000026635). Exon 10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Farp1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-91P8 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 10 starts from about 27.23% of the coding region. The knockout of Exon 10 will result in frameshift of the gene. The size of intron 9 for 5'-loxP site insertion: 1396 bp, and the size of intron 10 for 3'-loxP site insertion: 1182 bp. The size of effective cKO region: ~664 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 9 10 11 27 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Farp1 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(7164bp) | A(25.98% 1861) | C(23.1% 1655) | T(27.33% 1958) | G(23.59% 1690)

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% chr14 + 121235379 121238378 3000 browser details YourSeq 69 719 910 3000 91.4% chr13 - 12385542 12385845 304 browser details YourSeq 66 700 840 3000 90.0% chr4 - 111439194 111439398 205 browser details YourSeq 53 820 1360 3000 63.0% chr14 + 86543488 86543657 170 browser details YourSeq 53 720 838 3000 93.6% chr14 + 64573311 64573497 187 browser details YourSeq 51 715 840 3000 72.9% chr12 + 71803905 71803982 78 browser details YourSeq 49 706 843 3000 93.2% chr19 + 47924993 47925217 225 browser details YourSeq 48 700 843 3000 94.5% chr2 + 67602310 67602468 159 browser details YourSeq 41 728 844 3000 76.6% chr19 - 55263400 55263502 103 browser details YourSeq 40 715 788 3000 93.5% chr11 + 55265110 55265339 230 browser details YourSeq 39 700 798 3000 93.4% chr16 + 90905493 90905675 183 browser details YourSeq 38 720 843 3000 63.5% chr15 - 39815167 39815217 51 browser details YourSeq 37 753 842 3000 95.2% chr18 + 16431965 16432203 239 browser details YourSeq 36 679 734 3000 92.7% chr11 + 41632661 41632718 58 browser details YourSeq 35 781 843 3000 71.1% chr3 - 68569646 68569690 45 browser details YourSeq 35 784 839 3000 94.8% chr18 + 82537753 82538010 258 browser details YourSeq 33 782 843 3000 76.4% chr2 - 170431815 170431866 52 browser details YourSeq 33 720 856 3000 97.3% chr1 - 138700466 138700678 213 browser details YourSeq 30 715 759 3000 96.9% chr4 - 111438990 111439499 510 browser details YourSeq 27 807 844 3000 71.5% chr4 + 9143520 9143547 28

Note: The 3000 bp section upstream of Exon 10 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% chr14 + 121239043 121242042 3000 browser details YourSeq 164 1817 2021 3000 92.1% chr4 + 126477513 126477707 195 browser details YourSeq 162 1813 1996 3000 92.6% chr6 + 133958419 133958595 177 browser details YourSeq 157 1809 1995 3000 92.3% chr6 - 70543242 70543427 186 browser details YourSeq 154 1807 1996 3000 87.7% chr2 - 140434660 140434837 178 browser details YourSeq 153 1807 1996 3000 92.8% chr6 - 134705424 134705617 194 browser details YourSeq 152 1807 1996 3000 89.9% chr6 - 70632261 70632445 185 browser details YourSeq 151 1809 2001 3000 88.5% chr8 + 109549191 109549367 177 browser details YourSeq 151 1809 1995 3000 91.0% chr11 + 33577532 33577702 171 browser details YourSeq 150 1812 1997 3000 91.2% chr15 - 98944385 98944548 164 browser details YourSeq 148 1818 1997 3000 93.7% chr2 - 8117145 8117334 190 browser details YourSeq 148 1809 1994 3000 88.9% chr13 - 95504155 95504331 177 browser details YourSeq 148 1809 1977 3000 94.6% chr12 + 69234962 69235133 172 browser details YourSeq 147 1812 1988 3000 90.7% chr6 + 87474846 87475010 165 browser details YourSeq 146 1809 1992 3000 94.0% chr5 + 90295016 90295201 186 browser details YourSeq 146 1780 1959 3000 92.2% chr14 + 31100439 31100616 178 browser details YourSeq 146 1817 1993 3000 89.6% chr13 + 48247013 48247177 165 browser details YourSeq 145 1809 1996 3000 89.1% chr17 - 26145827 26145996 170 browser details YourSeq 145 1813 1992 3000 95.1% chr14 - 74658315 74658498 184 browser details YourSeq 145 1812 1992 3000 90.2% chr14 - 65312993 65313159 167

Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Farp1 FERM, RhoGEF (Arhgef) and pleckstrin domain protein 1 (chondrocyte-derived) [ Mus musculus (house mouse) ] Gene ID: 223254, updated on 12-Aug-2019

Gene summary

Official Symbol Farp1 provided by MGI Official Full Name FERM, RhoGEF (Arhgef) and pleckstrin domain protein 1 (chondrocyte-derived) provided by MGI Primary source MGI:MGI:2446173 See related Ensembl:ENSMUSG00000025555 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 Cdep; AW228844; BC030329 Expression Ubiquitous expression in limb E14.5 (RPKM 25.3), CNS E18 (RPKM 22.0) and 27 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 E5 See Farp1 in Genome Data Viewer

Exon count: 30

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (121035168..121283744)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (121434796..121682948)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 4 transcripts

Gene: Farp1 ENSMUSG00000025555

Description FERM, RhoGEF (Arhgef) and pleckstrin domain protein 1 (chondrocyte-derived) [Source:MGI Symbol;Acc:MGI:2446173] Gene Synonyms Cdep Location Chromosome 14: 121,035,200-121,283,744 forward strand. GRCm38:CM001007.2 About this gene This gene has 4 transcripts (splice variants), 252 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Farp1-201 ENSMUST00000026635.7 4885 1048aa ENSMUSP00000026635.6 Protein coding CCDS37015 F8VPU2 TSL:5 GENCODE basic APPRIS P1

Farp1-202 ENSMUST00000135010.7 797 211aa ENSMUSP00000116985.1 Protein coding - E9Q805 CDS 3' incomplete TSL:5

Farp1-203 ENSMUST00000137971.1 961 No protein - Retained intron - - TSL:3

Farp1-204 ENSMUST00000153607.1 777 No protein - lncRNA - - TSL:3

268.55 kb Forward strand

121.05Mb 121.10Mb 121.15Mb 121.20Mb 121.25Mb Genes Farp1-202 >protein coding B930095G15Rik-201 >lncRNA (Comprehensive set...

Farp1-201 >protein coding

Farp1-204 >lncRNA Farp1-203 >retained intron

Contigs < AC165163.2 AC167566.1 > < AC154618.2

Genes < Stk24-201protein coding (Comprehensive set...

Regulatory Build

121.05Mb 121.10Mb 121.15Mb 121.20Mb 121.25Mb Reverse strand 268.55 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: ENSMUST00000026635

248.18 kb Forward strand

Farp1-201 >protein coding

ENSMUSP00000026... MobiDB lite Low complexity (Seg) Superfamily Ubiquitin-like domain superfamily Dbl homology (DH) domain superfamily

SSF50729

FERM superfamily, second domain SMART Band 4.1 domain FERM adjacent (FA) Pleckstrin homology domain

FERM, C-terminal PH-like domain Dbl homology (DH) domain Prints Band 4.1 domain

Ezrin/radixin/moesin-like Pfam FERM, N-terminal FERM adjacent (FA) Dbl homology (DH) domain

FERM central domain Pleckstrin homology domain

FERM, C-terminal PH-like domain PROSITE profiles FERM domain Dbl homology (DH) domain Pleckstrin homology domain

PROSITE patterns FERM conserved site PANTHER PTHR45858:SF2

PTHR45858 Gene3D 3.10.20.90 PH-like domain superfamily

FERM/acyl-CoA-binding protein superfamily Dbl homology (DH) domain superfamily CDD cd17189 FERM central domain Dbl homology (DH) domain cd13235

FARP1/FARP2/FRMD7, FERM domain C-lobe cd01220

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 1048

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

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