Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox

Abstract

CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.

Biological warfare between two bacterial viruses in a genomic archipelago sheds light on the spread of CRISPR-Cas systems

“…According to the most recent classification of CRISPR-Cas systems, the I-Fa subtype has been classified as I-F2, and the I-Fb subtype as I-F1. However, as we have found here, subtype I-Fa presents the cas8f gene, contrary to what has been described for subtype I-F2 (12). Nevertheless, these differences in the established configurations of these defense systems are commonly found in virus-borne CRISPR-Cas systems (18).…”

“…CRISPR-Cas systems are highly diverse prokaryotic acquired immunity systems, currently classified into 2 classes, with multiple types and subtypes, which differ in the architecture of their components and their sequence specificity (12). They are composed of genes that are part of the different stages of this immune system and are generically called cas (CRISPR-associated genes) (13).…”

Compartmentalized CRISPR Reactions (CCR) for High-Throughput Screening of Guide RNA Potency and Specificity

“…CCR represents powerful yet accessible method to easily screen many candidate gRNAs (provided from the outputs of computational tools for gRNA design) for both activity and specificity, and as a way of rapidly identifying highly active and specific gRNA variants from large sets of gRNA/target/off-target combinations. We expect CCR can be applied to screen large sets ofgRNAs for other CRISPR effectors as well, particularly considering that the available computational tools for gRNA design are currently advanced for only a select few effectors, while new effectors with diverse properties are increasingly discovered [53].…”

Manipulating and studying gene function in human pluripotent stem cell models

“…Improvements in the performance of CRISPRa methodologies may come in many ways, first and foremost the development of novel effectors, such as CRISPRon. Strong potential lies within the growing number of type-II Cas9 and type-V Cas12 proteins [268], some of which may be coaxed into even more effective CRIS-PRa methods than SpCas9. Methods that recruit activator domains using RNA aptamers within the sgRNA, such as CRISPR-SAM, could prove advantageous to efficiently deliver CRISPRa effectors that are stably expressed in hPSCs and their derivatives.…”