CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems

Abstract

Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous recombination or nonhomologous end joining mechanisms can lead to the introduction of specific nucleotide substitutions or indel mutations, respectively. Furthermore, a nuclease-null Cas9 has been developed to regulate endogenous gene expression and to label genomic loci in living cells. Targeted genome editing and gene regulation mediated by Cas9 are easy to program, scale, and multiplex, allowing researchers to decipher the causal link between genetic and phenotypic variation. In this review, we describe the most notable applications of Cas9 in basic biology, translational medicine, synthetic biology, biotechnology, and other fields.

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

“…A short and conserved protospacer adjacent motif (PAM) sequence near the target site is required for the cleavage process of Cas9 [9,10]. CRISPR-Cas9 has been extensively used for genome editing in various cell types and organisms [11,12]. A series of structural studies of Streptococcus pyogenes Cas9 (SpyCas9) and its orthologs have revealed the detailed intermolecular interactions, as well as the conformational changes among different substrate-bound states [13][14][15][16][17][18].…”

Spatial gene drives and pushed genetic waves

“…http://dx.doi.org/10.1101/126722 doi: bioRxiv preprint first posted online Apr. 11, 2017; 2 The development of the CRISPR/Cas9 system [1][2][3][4], derived from an adaptive immune system in prokaryotes [5], has received much recent attention, in part due to its exceptional versatility as a gene editor in sexually-reproducing organisms, compared to similar exploitations of homologous recombination such as zinc-finger nucleases (ZFNs) and the TALENS system [4,6]. Part of the appeal is the potential for introducing a novel gene into a population, allowing control of highly pesticide-resistant crop pests and disease vectors such as mosquitoes [7][8][9][10].…”

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Genetic Engineering: Robotic Genetic Surgery

“…By using sgRNA guides, tumorigenic colon cancer genes and SNPs in coding as well as non-coding regions can be studied and potentially destroyed. In addition, colon carcinoma promoting proteins such as deregulated protein kinase C isozymes, MYC effector proteins, and various inflammatory promoters, are viable targets for the programmable nucleases [3, 28, 29]. By understanding the implications behind its uses as well as concerns, CRISPR/Cas9 stands at the forefront of biomedical advances and serves to change the landscape of genetic engineering in a broad spectrum of clinical malignancies.…”