Question

Predict how biological research will be transformed over the next decade by: 3rd generation sequencing, cyro-EM, and CRISPR-Cas9.

Answer 1

Biological research will be transformed over the next decade as follows:

1. 3rd generation sequencing:

A. Next generation sequencing (NGS) refers to the deep, high-throughput, in-parallel DNA sequencing technologies developed a few decades after the Sanger DNA sequencing method first emerged in 1977 and then dominated for three decades.

B. The NGS technologies are different from the Sanger method in that they provide massively parallel analysis, extremely high-throughput from multiple samples at much reduced cost.

C. Millions to billions of DNA nucleotides can be sequenced in parallel, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that were used with Sanger sequencing.

D. The second generation sequencing methods are characterized by the need to prepare amplified sequencing libraries before undertaking sequencing of the amplified DNA clones, whereas third-generation single molecular sequencing can be done without the need for creating the time-consuming and costly amplification libraries.

E. The parallelization of a high number of sequencing reactions by NGS was achieved by the miniaturization of sequencing reactions and, in some cases, the development of microfluidics and improved detection systems.

F. The time needed to generate the gigabase (Gb)-sized sequences by NGS was reduced from many years to only a few days or hours, with an accompanying massive price reduction.

G. The cost of sequencing the bacterial genome is now possible at about $1000 and the large-scale whole-genome sequencing (WGS) of 2,636 Icelanders.

H.Rapid progress in NGS technology and the simultaneous development of bioinformatics tools has allowed both small and large research groups to generate de novo draft genome sequences for any organism of interest.

I. The impact of NGS technology is indeed egalitarian in that it allows both small and large research groups the possibility to provide answers and solutions to many different problems and questions in the fields of genetics and biology, including those in medicine, agriculture, forensic science, virology, microbiology, and marine and plant biology.

2. Cyro-EM:

A. Over the pasts several years single-particle cryo-electron microscopy (cryo-EM) has emerged as a leading method for elucidating macromolecular structures at near-atomic resolution, rivaling even the established technique of X-ray crystallography.

B. Cryo-EM is now able to probe proteins as small as hemoglobin while avoiding the crystallization bottleneck entirely. The remarkable success of cryo-EM has called into question the continuing relevance of X-ray methods, particularly crystallography. To say that the future of structural biology is either cryo-EM or crystallography.

C. Crys-tallography remains better suited to yield precise atomic coordinates of macromolecules under a few hun-dred kDa in size, while the ability to probe larger, potentially more disordered assemblies is a distinct ad-vantage of cryo-EM.

D. Cryo-EM offers increasing insight into conformational and energy landscapes, particularly as algorithms to deconvolute conformational heterogeneity become more advanced.

E.  Cryo-EM uses the technique involves flash-freezing solutions of proteins or other biomolecules and then bombarding them with electrons to produce microscope images of individual molecules.

F. Ultimatelyt future of both techniques depends on how their individual strengths are utilized to tackle questions on the frontiers of structural biology. Structure determination is just one piece of a much larger puzzle: a central challenge of modern structural biology is to relate structural information to biological function. In this perspective, we share insight from several leaders in the field and examine the unique and complementary ways in which X-ray methods and cryo-EM can shape the future of structural biology.

3. CRISPR-Cas9: A. CRISPR/Cas9 is an RNA guided endonuclease targeting the DNA.

B. CRISPR/Cas9 has high efficiency, accuracy, and ease of use for GE.

C.CRISPR/Cas9 system has been the best choice for GE, but despite its extensive use and applications, there are still some limitations to its more widespread application.

D. Genome editing (GE) has revolutionized biological research through the new ability to precisely edit the genomes of living organisms.

E.The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has widely been used in GE due to its high efficiency, ease of use, and accuracy. It can be used to add desirable and remove undesirable alleles simultaneously in a single event.

F. we discuss various applications of CRISPR/Cas9 in a range of important crops, compare it with other GE tools, and review its mechanism, limitations, and future possibilities. Various newly emerging CRISPR/Cas systems.

G. CRISPR is a tool used by researchers to precisely edit genes and has shown potential for treating genetic diseases.
H. CRISPR genome editing is a promising field that enables researchers to precisely delete, replace or edit genes.
I. CRISPR-Cas is a prokaryotic defence system whereby bacteria use RNA molecules and CRISPR-associated (Cas) proteins to target and destroy the DNA of invading viruses. This molecular machinery has been repurposed by researchers to target and edit specific sections of any DNA, whether bacterial or human.
J. many researchers have sought improvements to CRISPR with the gene editing method expected to continue development well into the future.

K. Multiplex CRISPR systems to target lots of genes, researchers at ETH Zurich in Switzerland swapped the Cas9 enzyme for Cas12a. Using this plasmid allowed the researchers to simultaneously edit genes in 25 target sites. The team predicts that dozens or even hundreds more sites could be modified using this method.