A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

Content introduction:

  • Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase
  • De novo domestication of wild tomato using genome editing
  • A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes
  • Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A
  • Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

1. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

Here Emily K Schutsky at University of Pennsylvania in Philadelphia, Pennsylvania, USA and his colleagues present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, they found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

Read more, please click https://www.nature.com/articles/nbt.4204

2. De novo domestication of wild tomato using genome editing

Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost. We devised a CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. Agustin Zsögön at Universidade Federal de Viçosa in Viçosa, Brazil and his colleagues report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, their engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Their results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants.

Read more, please click https://www.nature.com/articles/nbt.4272

3. A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. Kyros Kyrou at Imperial College in London, UK and his colleagues found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.

Read more, please click https://www.nature.com/articles/nbt.4245

4. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Base editors (BEs) have been used to create C-to-T substitutions in various organisms. However, editing with rat APOBEC1-based BE3 is limited to a 5-nt sequence editing window and is inefficient in GC contexts. Here, Yuan Zong at Chinese Academy of Sciences in Beijing, China and his colleagues show that a base editor fusion protein composed of Cas9 nickase and human APOBEC3A (A3A-PBE) converts cytidine to thymidine efficiently in wheat, rice and potato with a 17-nucleotide editing window at all examined sites, independent of sequence context.

Read more, please click https://www.nature.com/articles/nbt.4261

5. Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell’s identity and its position in a tissue. Keren Bahar Halpern at Weizmann Institute of Science in Rehovot, Israel and his colleagues recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here they used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs’ tissue coordinates. They applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, they reconstructed LEC zonation and extracted a landmark gene panel that they used to spatially map LEC scRNAseq data. Their approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.

Read more, please click https://www.nature.com/articles/nbt.4231

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