Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

Background: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. Results: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Conclusions: Our results provide a technical platform for high-throughput knock-in

CLICK:One-step generation of conditional knockout mice

Abstract Background CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. Results Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. Conclusions The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice

Schematic models of the flowering pathway in Nona Bokra and Koshihikari under ND and LD conditions.

<p><i>RFT1</i> in Nona Bokra is defective, but the pathway that represses <i>Hd3a</i> expression is functional. Koshihikari has functional <i>RFT1</i>, but has defects (in <i>Hd6</i> and <i>Hd16</i>) in the pathway that represses <i>Hd3a</i>. Under ND conditions, <i>RFT1</i> begin to express from summer, but it cannot induce <i>Hd3a</i> expression in Nona Bokra. In autumn (short day conditions), <i>Hd3a</i> expression is induced and Nona Bokra can flower. Under LD conditions, <i>RFT1</i> expression does not occur, and <i>Hd3a</i> is repressed by flowering repressor genes, and Nona Bokra cannot flower. In Koshihikari, <i>Hd3a</i> and <i>RFT1</i> express under LD and ND condition, and it shows early flowering. Geographic location of the original cultivation regions of Koshihikari and Nona Bokra is shown on the right.</p

Additional file 1: of Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

The following additional data are available with the online version of this paper. Additional data file 1 contains the figures including IDA analysis of PITCh-donor, PCR screenings of mice, sequencing of non-knock-in Actb alleles, FACS and LSM analysis of human cells, Exo1 western blotting, Exo1 toxicity analysis, off-target analysis in mice, germline transmission, linear PCR donor injection, and sequence alignments of hACTB and its off-target sites, and tables of these results and a list of the oligo DNAs and RNAs used in this study. (DOCX 14447 kb

Natural Variation of the <i>RICE FLOWERING LOCUS T 1</i> Contributes to Flowering Time Divergence in Rice

<div><p>In rice (<i>Oryza sativa</i> L.), there is a diversity in flowering time that is strictly genetically regulated. Some <i>indica</i> cultivars show extremely late flowering under long-day conditions, but little is known about the gene(s) involved. Here, we demonstrate that functional defects in the florigen gene <i>RFT1</i> are the main cause of late flowering in an <i>indica</i> cultivar, Nona Bokra. Mapping and complementation studies revealed that sequence polymorphisms in the <i>RFT1</i> regulatory and coding regions are likely to cause late flowering under long-day conditions. We detected polymorphisms in the promoter region that lead to reduced expression levels of <i>RFT1</i>. We also identified an amino acid substitution (E105K) that leads to a functional defect in Nona Bokra RFT1. Sequencing of the <i>RFT1</i> region in rice accessions from a global collection showed that the E105K mutation is found only in <i>indica</i>, and indicated a strong association between the <i>RFT1</i> haplotype and extremely late flowering in a functional <i>Hd1</i> background. Furthermore, SNPs in the regulatory region of <i>RFT1</i> and the E105K substitution in 1,397 accessions show strong linkage disequilibrium with a flowering time–associated SNP. Although the defective E105K allele of <i>RFT1</i> (but not of another florigen gene, <i>Hd3a</i>) is found in many cultivars, relative rate tests revealed no evidence for differential rate of evolution of these genes. The ratios of nonsynonymous to synonymous substitutions suggest that the E105K mutation resulting in the defect in <i>RFT1</i> occurred relatively recently. These findings indicate that natural mutations in <i>RFT1</i> provide flowering time divergence under long-day conditions.</p> </div

mRNA levels of <i>Ehd1</i>, <i>RFT1</i> and <i>Hd3a</i> in control, #3098-2-1-1 and Nona Bokra growing under LD conditions.

<p>Graphical genotypes around <i>RFT1</i> and <i>Hd3a</i> are shown below (for graphical genotypes of the whole genomes, see Figure S1). White and black boxes represent Koshihikari and Nona Bokra origin, respectively. All lines have functional <i>Hd1</i> and <i>Hd16</i>. <i>Hd1*</i> indicates strong allele [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075959#B52" target="_blank">52</a>]. <i>Ehd1</i>, <i>RFT1</i> and <i>Hd3a</i> expression was below the detection limit in Nona Bokra.</p

Flowering time of plants from the core collection under SD, LD and ND conditions.

<p>Accessions were divided into four groups by <i>RFT1</i> haplotypes and <i>Hd1</i> function (functional <i>Hd1</i> is shown in bold). <i>RFT1</i> haplotypes were divided into two groups, candidate functional (groups I, III and IV) or defective (group II) haplotypes. Accessions were grouped according to flowering time (in 10-day steps). The diameter of the black circles reflects the number of accessions. BIN, Bingala; BKH, Bei Khei; RGS, Radin Goi Sesat. The distributions of flowering time of group II with functional <i>Hd1</i> deviated from other distributions under LD and ND conditions (<i>P</i><0.01, Kolmogorov-Smirnov test), but not under SD conditions (<i>P</i> = 0.135).</p

Amino acid sequences of RFT1 and Hd3a and phenotypes of RFT1 overexpressors.

<p>(A) Comparison of amino acid sequences of rice and Arabidopsis FT-like proteins. Rice RFT1 and Hd3a (from three cultivars as indicated), and Arabidopsis FT, TSF and TFL1 are shown. Conserved amino acids are shaded in black, dark gray or light gray depending on the level of conservation. The blue and red arrows indicate the V33A and E105K sites, respectively. The red boxes indicate the binding interface with 14-3-3 protein [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075959#B57" target="_blank">57</a>]. (B) 35S:<i>RFT1</i> constructs. <i>RFT1</i> alleles used: Nip, Nipponbare; Kasa, Kasalath; NB, Nona Bokra. E105K, Nipponbare allele with the introduced point mutation. Black and gray boxes denote ORF and UTR, respectively. Amino acids differing from those in Nipponbare are shown as white letters. (C) Regenerated plants (cv. Nipponbare) transformed with 35S:<i>RFT1</i> constructs or with the empty vector (Vec). Bar = 1 cm. (D, E) 35S:<i>nipRFT1</i> (D) and 35S:<i>kasaRFT1</i> (E) plants at higher magnifications. Bar = 2 mm. (F) Flowering time of 20 T₀ plants overexpressing each of the 35S:<i>RFT1</i> constructs or vector control under LL (continuous light) and LD conditions. Day 0 corresponds to the date of transplanting onto regeneration media under LL conditions. On day 20, counting of the flowering plants was started, and plates were transferred to LD. Each open ellipse indicates an individual plant. (G) Expression levels of <i>RFT1</i>, <i>Hd3a</i>, <i>FTL</i> and <i>Hd1</i> expression in T₀ plants transformed with the 35S:<i>RFT1</i> constructs or vector under LL conditions. N.D. indicates that transcripts were not detected. Samples were taken 20 days after transplanting onto regeneration media. Samples from five plants were mixed together. Each bar represents the mean ± SD (technical replicates n=3). Expression values were plotted on a log₁₀ scale. <i>Ehd1</i> was not detected (data not shown).</p

Genetic complementation test and effect of genetic background.

<p>(A, B) Flowering time of control (Con, see Figure S1), SL520 (A) or Nona Bokra (B), and T₂ plants harboring the empty vector (+Vec) or the Koshihikari <i>RFT1</i> genomic fragment (+<i>KosRFT1</i> 5 kb; see Figure 1D) in SL520 (A) or Nona Bokra (B) under LD conditions. Each bar represents the mean ± SD (n = 3–5). (C) Genetic complementation test using F₃ lines derived from a cross between Koshihikari and SL520 (Figure S1) under LD conditions. Graphical genotypes of <i>RFT1</i>/<i>Hd3a</i> regions are indicated. Lines 3098#2 and 3095#3 have Nona Bokra and Koshihikari <i>Hd3a</i> homozygous alleles, respectively. For transgenic lines (T₀), each bar represents an individual plant. Control and genotypes of other regions are shown in Figure S1.</p

The mRNA levels of <i>RFT1</i> and <i>Hd3a</i> in Koshihikari, Nipponbare and Nona Bokra during the growing period in a natural field.

<p>(A) Expression levels of <i>RFT1</i> and <i>Hd3a</i> in top leaves. Expression values were plotted on a log₁₀ scale. Black triangles indicate the flowering date for each cultivar. (B) Changes in daylength at the growing location. Open triangle indicates the sowing date for each cultivar. Yellow boxes represent the period of long-day conditions (>13 h daylength).</p