CRISPR with a Happy Ending: Non‐Templated DNA Repair for Prokaryotic Genome Engineering

Abstract

The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR‐Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR‐Cas and other nucleases mediate double‐strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template‐independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non‐homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.

Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

“…coli engineering 38 , does not operate efficiently in Pseudomonas 39 —likely due to limited activity of phage recombinases in these species, or because native proteins interfere with their action. While CRISPR/Cas toolsets accelerated microbial engineering efforts 40 , 41 , non-homologous end joining (NHEJ) mechanisms are virtually absent in prokaryotes 42 , preventing the introduction of frameshifts in target loci—and functional knock-outs thereof. A recent proof-of-principle study on base editing in Pseudomonas by Chen et al 41 .…”

Large scale screening of CRISPR guide RNAs using an optimized high throughput robotics system

“…Improved methods of predicting amino acid structure and function have allowed investigators to continue identifying CRISPR-Cas systems from new organisms, therefore growing the field and increasing potential for biotechnological innovation. Some examples include applications in microbial engineering 2 , 3 , agricultural engineering 4 , 5 , novel analyte detection 6 , disease-mediation 7 , stem cell development 8 and viral detection 9 . CRISPR-Cas systems generally fall into two classes based on the number of required Cas components for full function, wherein Class 2 (containing types II, V, and VI) consists of single protein systems that are most useful for biotechnological development due to their relative simplicity 10 .…”

CRISPR/Cas12a‐mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus

“…As CRISPR/Cas12a creates a staggered end resulting a 5‐nt 5′ overhang rather than a blunt cleavage product, which might promote the double‐strand break repair mediated by non‐homologous end joining (NHEJ) and/or microhomology‐mediated end joining (MMEJ) mechanism (Finger‐Bou et al., 2020 ). The initial studies revealed that about 30%˜40% editing efficiency was obtained when targeting ccoO or nifH gene, which was not satisfactory when compared to NHEJ‐mediated CRISPR/Cas in Streptomyces coelicolor (Li et al., 2018 ) and E. coli (Zheng et al., 2017 ).…”