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Mouse Fcrla Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Fcrla gene (NCBI Reference Sequence: NM_001160215 ; Ensembl: ENSMUSG00000038421 ) is located on Mouse chromosome 1. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000046322). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fcrla gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-100H5 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 targeted allele exhibit largely normal T-dependent and T-independent antibody responses with an increase in IgG1 after secondary challenge with sheep red blood cells.

Exon 2~4 is not frameshift exon, and covers 69.69% of the coding region. The size of intron 1 for 5'-loxP site insertion: 5054 bp, and the size of intron 4 for 3'-loxP site insertion: 2439 bp. The size of effective cKO region: ~2074 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 5 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Fcrla 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(8574bp) | A(28.53% 2446) | C(22.01% 1887) | T(26.0% 2229) | G(23.47% 2012)

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% chr1 - 170922658 170925657 3000 browser details YourSeq 451 1 493 3000 96.0% chr5 + 137615614 137616108 495 browser details YourSeq 440 1 488 3000 95.7% chr14 + 12867540 12868030 491 browser details YourSeq 432 32 481 3000 98.3% chr13 - 66900617 66902802 2186 browser details YourSeq 414 1 489 3000 91.7% chr10 + 75767148 75767627 480 browser details YourSeq 351 1 436 3000 90.4% chr17 - 23161489 23161925 437 browser details YourSeq 268 1592 2004 3000 83.7% chr13 + 50988205 50988622 418 browser details YourSeq 253 1603 2002 3000 87.4% chr5 + 76670705 76671104 400 browser details YourSeq 251 1599 1978 3000 85.0% chr9 + 30366026 30366403 378 browser details YourSeq 247 1599 2002 3000 82.7% chr3 + 129853762 129854166 405 browser details YourSeq 244 1599 2002 3000 81.5% chrX + 101625063 101625464 402 browser details YourSeq 243 1599 1983 3000 83.9% chr10 - 89273238 89273622 385 browser details YourSeq 240 1598 2002 3000 79.7% chr10 - 10982897 10983291 395 browser details YourSeq 239 1598 2002 3000 85.4% chr2 - 105417973 105418380 408 browser details YourSeq 238 1442 2002 3000 85.8% chr14 - 53291514 53292287 774 browser details YourSeq 237 1587 1973 3000 84.3% chr4 + 89121476 89121859 384 browser details YourSeq 236 1599 2000 3000 81.6% chr1 - 175583388 175583780 393 browser details YourSeq 233 1599 2002 3000 81.1% chr8 - 66725804 66726202 399 browser details YourSeq 233 1599 2002 3000 86.4% chr12 - 27861483 27861899 417 browser details YourSeq 232 1599 1999 3000 86.2% chr2 + 112137584 112137968 385

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% chr1 - 170917584 170920583 3000 browser details YourSeq 529 212 765 3000 97.9% chr13 - 119673621 119674179 559 browser details YourSeq 529 214 774 3000 97.5% chr18 + 40342780 40343346 567 browser details YourSeq 528 212 766 3000 97.7% chrX + 70624941 70625500 560 browser details YourSeq 527 212 765 3000 97.7% chr19 - 8249918 8250476 559 browser details YourSeq 527 212 765 3000 97.7% chr12 - 20492928 20493486 559 browser details YourSeq 527 212 765 3000 97.7% chr2 + 133228428 133228986 559 browser details YourSeq 525 212 765 3000 97.5% chr10 + 35972231 35972788 558 browser details YourSeq 524 213 765 3000 97.5% chr10 - 129510492 129511049 558 browser details YourSeq 524 214 765 3000 97.7% chrX + 122245654 122246275 622 browser details YourSeq 524 213 765 3000 97.5% chr19 + 42557232 42557789 558 browser details YourSeq 523 220 765 3000 98.0% chr12 + 21695972 21696522 551 browser details YourSeq 522 212 768 3000 97.0% chr6 - 147459955 147460516 562 browser details YourSeq 522 212 765 3000 97.7% chr6 + 64331746 64332306 561 browser details YourSeq 520 212 771 3000 96.6% chr2 - 4656667 4657228 562 browser details YourSeq 519 220 765 3000 97.9% chr6 + 68904207 69328101 423895 browser details YourSeq 517 212 765 3000 96.8% chr8 - 30164511 30165069 559 browser details YourSeq 516 212 765 3000 97.1% chr19 + 13159808 13160368 561 browser details YourSeq 515 212 765 3000 96.6% chr13 - 83193377 83193935 559 browser details YourSeq 515 215 766 3000 97.0% chr8 + 65911474 65912030 557

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Fcrla Fc receptor-like A [ Mus musculus (house mouse) ] Gene ID: 98752, updated on 12-Aug-2019

Gene summary

Official Symbol Fcrla provided by MGI Official Full Name Fc receptor-like A provided by MGI Primary source MGI:MGI:2138647 See related Ensembl:ENSMUSG00000038421 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 FCRL; FREB; Fcrx; FCRL1; Freb1; mFREB; mFcrX; Fcrlm1; BB219290 Expression Biased expression in spleen adult (RPKM 43.6), mammary gland adult (RPKM 10.0) and 3 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 H3 See Fcrla in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (170917594..170932216, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (172847725..172857714, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 6 transcripts

Gene: Fcrla ENSMUSG00000038421

Description Fc receptor-like A [Source:MGI Symbol;Acc:MGI:2138647] Gene Synonyms FCRL1, FREB, Fcrx, Freb1, mFREB, mFcrX Location Chromosome 1: 170,917,576-170,927,583 reverse strand. GRCm38:CM000994.2 About this gene This gene has 6 transcripts (splice variants), 129 orthologues, 13 paralogues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fcrla- ENSMUST00000046322.13 1679 353aa ENSMUSP00000036380.7 Protein coding CCDS48440 Q920A9 TSL:1 201 GENCODE basic APPRIS P4

Fcrla- ENSMUST00000159171.1 1371 352aa ENSMUSP00000124853.1 Protein coding CCDS48439 Q920A9 TSL:1 203 GENCODE basic APPRIS ALT2

Fcrla- ENSMUST00000162136.1 663 216aa ENSMUSP00000124859.1 Protein coding - E0CX78 CDS 3' 205 incomplete TSL:3

Fcrla- ENSMUST00000162887.1 509 79aa ENSMUSP00000124469.1 Protein coding - E0CY58 CDS 3' 206 incomplete TSL:2

Fcrla- ENSMUST00000159149.7 1116 177aa ENSMUSP00000125074.1 Nonsense mediated - F7DEW9 CDS 5' 202 decay incomplete TSL:5

Fcrla- ENSMUST00000161050.1 460 No - lncRNA - - TSL:3 204 protein

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30.01 kb Forward strand 170.91Mb 170.92Mb 170.93Mb Genes Gm37030-201 >processed pseudogene (Comprehensive set...

Contigs < AC113490.10 < AC115959.17 Genes (Comprehensive set... < Fcrlb-201protein coding < Fcrla-201protein coding

< Fcrla-202nonsense mediated decay < Gm2962-201processed pseudogene

< Fcrla-203protein coding

< Fcrla-205protein coding

< Fcrla-206protein coding

< Fcrla-204lncRNA

Regulatory Build

170.91Mb 170.92Mb 170.93Mb Reverse strand 30.01 kb

Regulation Legend Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript pseudogene RNA gene

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Transcript: ENSMUST00000046322

< Fcrla-201protein coding

Reverse strand 10.01 kb

ENSMUSP00000036... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin subtype 2

Immunoglobulin subtype Pfam Immunoglobulin-like domain PF13927

PROSITE profiles Immunoglobulin-like domain PANTHER PTHR11481:SF71

PTHR11481 Gene3D Immunoglobulin-like fold

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 353

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

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