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1. Modeling the efficacy of CRISPR gene drive for snail immunity on schistosomiasis control

   https://doi.org/10.1371/journal.pntd.0010894  

   Grewelle, Richard E.; Perez-Saez, Javier; Tycko, Josh; Namigai, Erica K.; Rickards, Chloe G.; De Leo, Giulio A. (October 2022, PLOS Neglected Tropical Diseases)

   Rinaldi, Gabriel (Ed.)

   CRISPR gene drives could revolutionize the control of infectious diseases by accelerating the spread of engineered traits that limit parasite transmission in wild populations. Gene drive technology in mollusks has received little attention despite the role of freshwater snails as hosts of parasitic flukes causing 200 million annual cases of schistosomiasis. A successful drive in snails must overcome self-fertilization, a common feature of host snails which could prevents a drive’s spread. Here we developed a novel population genetic model accounting for snails’ mixed mating and population dynamics, susceptibility to parasite infection regulated by multiple alleles, fitness differences between genotypes, and a range of drive characteristics. We integrated this model with an epidemiological model of schistosomiasis transmission to show that a snail population modification drive targeting immunity to infection can be hindered by a variety of biological and ecological factors; yet under a range of conditions, disease reduction achieved by chemotherapy treatment of the human population can be maintained with a drive. Alone a drive modifying snail immunity could achieve significant disease reduction in humans several years after release. These results indicate that gene drives, in coordination with existing public health measures, may become a useful tool to reduce schistosomiasis burden in selected transmission settings with effective CRISPR construct design and evaluation of the genetic and ecological landscape. 

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2. Gene flow accelerates adaptation to a parasite

   https://doi.org/10.1093/evolut/qpad048  

   Lewis, Jordan A.; Kandala, Prathyusha; Penley, McKenna J.; Morran, Levi T. (March 2023, Evolution)

   Abstract Gene flow into populations can increase additive genetic variation and introduce novel beneficial alleles, thus facilitating adaptation. However, gene flow may also impede adaptation by disrupting beneficial genotypes, introducing deleterious alleles, or creating novel dominant negative interactions. While theory and fieldwork have provided insight into the effects of gene flow, direct experimental tests are rare. Here, we evaluated the effects of gene flow on adaptation in the nematode Caenorhabditis elegans during exposure to the bacterial parasite, Serratia marcescens. We evolved hosts against nonevolving parasites for 10 passages while controlling host gene flow and source population. We used source nematode populations with three different genetic backgrounds (one similar to the sink population and two different) and two evolutionary histories (previously adapted to S. marcescens or naive). We found that populations with gene flow exhibited greater increases in parasite resistance than those without gene flow. Additionally, gene flow from adapted populations resulted in greater increases in resistance than gene flow from naive populations, particularly with gene flow from novel genetic backgrounds. Overall, this work demonstrates that gene flow can facilitate adaptation and suggests that the genetic architecture and evolutionary history of source populations can alter the sink population’s response to selection. 

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3. Non‐self recognition‐based self‐incompatibility can alternatively promote or prevent introgression

   https://doi.org/10.1111/nph.17249  

   Harkness, Alexander; Brandvain, Yaniv (February 2021, New Phytologist)

   Summary Self‐incompatibility alleles (S‐alleles), which prevent self‐fertilisation in plants, have historically been expected to benefit from negative frequency‐dependent selection and invade when introduced to a new population through gene flow. However, the most taxonomically widespread form of self‐incompatibility, the ribonuclease‐based system ancestral to the core eudicots, functions through collaborative non‐self recognition, which can affect both short‐term patterns of gene flow and the long‐term process of S‐allele diversification.We analysed a model of S‐allele evolution in two populations connected by migration, focussing on comparisons among the fates of S‐alleles initially unique to each population and those shared among populations.We found that both shared and unique S‐alleles from the population with more unique S‐alleles were usually fitter compared with S‐alleles from the population with fewer S‐alleles. Resident S‐alleles often became extinct and were replaced by migrant S‐alleles, although this outcome could be averted by pollen limitation or biased migration.Collaborative non‐self recognition will usually either result in the whole‐sale replacement of S‐alleles from one population with those from another or else disfavour introgression of S‐alleles altogether. 

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4. Efficient protein tagging and cis -regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants

   https://doi.org/10.1093/plcell/koad139  

   Kumar, Jitesh; Char, Si Nian; Weiss, Trevor; Liu, Hua; Liu, Bo; Yang, Bing; Zhang, Feng (May 2023, The Plant Cell)

   Abstract Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5′ overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5′ overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner. 

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5. Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

   https://doi.org/10.1002/pld3.168  

   Mallett, Darren R.; Chang, Mingqin; Cheng, Xiaohang; Bezanilla, Magdalena (September 2019, Plant Direct)

   Abstract CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping. 

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