Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

Although the application of CRISPR/Cas9 genome engineering approaches was first reported in apicomplexan parasites only 3 years ago, this technology has rapidly become an essential component of research on apicomplexan parasites. This review briefly describes the history of CRISPR/Cas9 and the principles behind its use along with documenting its implementation in apicomplexan parasites, especially Plasmodium spp. and Toxoplasma gondii. We also discuss the recent use of CRISPR/Cas9 for whole genome screening of gene knockout mutants in T. gondii and highlight its use for seminal genetic manipulations of Cryptosporidium spp. Finally, we consider new variations of CRISPR/Cas9 that have yet to be implemented in apicomplexans. Whereas CRISPR/Cas9 has already accelerated rapid interrogation of gene function in apicomplexans, the full potential of this technology is yet to be realized as new variations and innovations are integrated into the field.

In recent years, stimuli responsive polymer based gene delivery vehicle design for cancer treatment and treatment of other genetic disorders has received extensive attention. Early studies focusing on DNA delivery have been facilitated by functional polymers and this area has seen further growth spurred by recent gene silencing strategies developed for small RNA [i.e., small interfering RNA (siRNA) or micro RNA (miRNA)] delivery. DNA and small RNAs possess analogous properties; however, their explicit differences define the specific challenges associated with the delivery route and the design of functional materials to overcome distinct challenges. Apart from classical gene delivery, the recent advances in genome editing have revealed the necessity of new delivery devices for genome editing tools. A system involving CRISPR (clustered, regularly interspaced, short palindromic repeats) and an endonuclease CRISPR-associated protein 9 (Cas9) coupled with a short, single-guide RNA (sgRNA) has emerged as a promising tool for genome editing along with functional delivery systems. For all these nucleic acid based treatments, the internal or external physiochemical changes in the biological tissue/cells play a major role in the design of stimuli responsive delivery materials for both in vitro and in vivo applications. This review emphasizes the recent advances in the use of pH, temperature, and redox potential-responsive polymers overcoming hurdles for delivery of gene and gene editing tools for both in vitro and in vivo applications. Specifically the chapter focuses on recently proposed delivery strategies, types of delivery systems, and polymer synthesis/modification methods. The recent advances in CRISPR/Cas9-sgRNA technology and delivery are also described in a separate section. The review ends with current clinical trials, concluding remarks, and future perspectives.

Advances in embryology, genetics, and regenerative medicine regularly attract attention from scientists, scholars, journalists, and policymakers, yet implications of these advances may be broader than commonly supposed. Laboratories culturing human embryos, editing human genes, and creating human-animal chimeras have been working along lines that are now becoming intertwined. Embryogenic methods are weaving traditional in vivo and in vitro distinctions into a new “in vivitro” (in life in glass) fabric. These and other methods known to be in use or thought to be in development promise soon to bring society to startling choices and discomfiting predicaments, all in a global effort to supply reliably rejuvenating stem cells, to grow immunologically nonprovocative replacement organs, and to prevent, treat, cure, or even someday eradicate diseases having genetic or epigenetic mechanisms. With humanity’s human-engineering era now begun, procedural prohibitions, funding restrictions, institutional controls, and transparency rules are proving ineffective, and business incentives are migrating into the most basic life-sciences inquiries, wherein lie huge biomedical potentials and bioethical risks. Rights, health, and heritage are coming into play with bioethical presumptions and formal protections urgently needing reassessment.

Transgenesis for Strongyloides and Parastrongyloides was accomplished in 2006 and is based on techniques derived for Caenorhabditis elegans over two decades earlier. Adaptation of these techniques has been possible because Strongyloides and related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. In Strongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes in S. stercoralis requires their integration into the genome, and stable lines have been derived from integrants created using the piggyBac transposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis in Strongyloides and Parastrongyloides opens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.