Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants

正Dear Editor,CRISPR (clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants. The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA (sgRNA) incorporating a target sequence (Li et al., 2019). A Cas9/sgRNA complex re-     

Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco

Plant Molecular Biology - Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered...     

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.     

The trouble with collective nouns for genome editing

Mammalian Genome - We should start as we mean to go on and try to avoid the confusion most of us experience when bombarded with acronyms with overstated significations. You will be familiar with...     

Current and future editing reagent delivery systems for plant genome editing

Many genome editing tools have been developed and new ones are anticipated; some have been extensively applied in plant genetics, biotechnology and breeding, especially the CRISPR/Cas9 system. These technologies have opened up a new era for crop improvement due to their precise editing of user-specified sequences related to agronomic traits. In this review, we will focus on an update of recent developments in the methodologies of editing reagent delivery, and consider the pros and cons of current delivery systems. Finally, we will reflect on possible future directions.     

Erratum to: Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

正The same figure was misused for the PCR/RE assay results of Gn1a and GW2 fragments in Figure 3, and the arrows in the graphicsal result of GW2 were not on the tape. The corrected Figure 3 is as follows.     

Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice

正Most of the important agronomic traits in crop plants,such as yield,quality and stress response,are quantitative and jointly controlled by many genomic loci or major genes.Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci(QTLs).However,the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs.In certain cases,this approach might     

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.     

Targeted genome editing of plants and plant cells for biomanufacturing

Plants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as “plant molecular farming” (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose “chassis” for PMF.

     

Applications and potential of genome editing in crop improvement

Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism’s genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.     

A genome screen of multiplex sibships with prostate cancer

Analysis of a genome screen of 504 brothers with prostate cancer (CaP) who were from 230 multiplex sibships identified five regions with nominally positive linkage signals, on chromosomes 2q, 12p, 15q, 16p, and 16q. The strongest signal in these data is found on chromosome 16q, between markers D16S515 and D16S3040, a region suspected to contain a tumor-suppressor gene. On the basis of findings from previous genome screens of families with CaP, three preplanned subanalyses were carried out, in the hope of increasing the subgroup homogeneity. Subgroups were formed by dividing the sibships into a group with a positive family history (FH+) that met criteria for "hereditary" CaP (n=111) versus those which did not meet the criteria (n=119) and by dividing the families into those with a mean onset age below the median (n=115) versus those with a mean onset age above the median (n=115). A separate subanalysis was carried out for families with a history of breast cancer (CaB+ [n=53]). Analyses of these subgroups revealed a number of potentially important differences in regions that were nonsignificant when all the families were analyzed together. In particular, the subgroup without a positive family history (FH-) had a signal in a region that is proximal to the putative site of the HPC1 locus on chromosome 1, whereas the late-age-at-onset group had a signal on 4q. The CaB+ subgroup revealed a strong linkage signal at 1p35.1.     

Expanding the scope of genome editing with SpG and SpRY variants in rice

正Dear Editor,The clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system, since it was excavated, has been rapidly developed and sparked a revolution in the genome editing field. In principle,CRISPR/Cas9 system relies on the recognition of specific loci on the genome, which is titled the protospacer adjacent motif (PAM). However, the canonical Streptococcus pyogenes Cas9 (SpCas9) nuclease only recognizes NGG or NAG PAMs, rendering an inherent obstacle in amplifying the application of CRISPR/Cas9 technology.     

Putative homozygous mutations in regenerated plants of rice

Summary Both normal and putative homozygous mutant (dwarf mutant) rice plants were regenerated from diploid seed callus, cultured in the presence of 1% NaCl. This trait was transmitted at least through the eighth genration (D₈) of regenerated plants (D₁) by self-pollination, as a homozygous mutation. However, the trait disappeared in the F₁, F₂, F₃ and F₄ obtained by reciprocal crosses of mutant plants with either control plants or with progeny of normal regenerated plants. Chimeric reversion of the homozygous mutant trait was observed and the revertant phenotype was transmitted stably to at least three successive generations. Similar dwarf types of homzygous mutation were observed independently in the two varieties, Norin 8 and Nipponbare, in an experimental series of ca. 3000 D₁ plants. The frequency of mutations among regenerated plants was calculated to be 1.8×10^-2. The mechanism responsible for these phenomena may be heritable gene inactivation induced by in vitro culture.     

Novel reporter systems for facile evaluation of I-SceI-mediated genome editing

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects.     

Epileptic seizures in rats homozygous for two mutations, zitter and tremor

In the rat, zitter (zi) and tremor (tm) are autosomal recessive mutations that in homozygotes caused curled whisker hair, and tremor when the animals move. In both mutant types, the presence of many vacuoles gives the central nervous system a spongy appearance. The mutants differ sharply in one important characteristic: zitter rats are fertile, tremor rats are sterile. The F1 hybrids between zitter homozygous females and a tremor heterozygous male were all normal, and data from subsequent crosses revealed that zi and tm are neither allelic nor linked to each other. In animals homozygous for both mutations, an epileptic seizure frequently and spontaneously occurred from 7 or 8 weeks of age. Although the epileptic seizure has not been studied in detail, it may be an excellent animal model of epilepsy.