Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Abstract

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

“…Additionally, the target site must contain a proto-spacer adjacent motif (PAM) immediately adjacent to the 20 nt target site. For Streptococcus pyogenes Cas9, this PAM motif is 5’-NGG-3’; however, 5’-NAG-3’ motifs may also occasionally be recognized ( Thompson et al, 2021 ) ( Table 2 ). Since there are many potential PAM motifs within a genome, Cas9:sgRNA complexes can only spend a short amount of time (<30 ms) at any given PAM motif ( Jones et al, 2017 ).…”

“…Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems function as an adaptive immune system against foreign nucleic acids in archaea and bacteria ( Marraffini and Sontheimer, 2010 ; Rath et al, 2015 ; Jiang and Doudna, 2017 ; Koonin et al, 2017 ; Thompson et al, 2021 ). Using a dual RNA-guided CRISPR endonuclease, such a Streptococcus pyogenes Cas9 (spCas9), prokaryotic organisms can specifically recognize and cleave invading foreign DNA ( Jiang and Doudna, 2017 ; Thompson et al, 2021 ). Crucially, the ability of Cas proteins to target, bind, and cleave selected nucleic acid sequences has been exploited for precise genome editing of eukaryotic organisms ( Jinek et al, 2012 ; Jiang and Doudna, 2017 ; Thompson et al, 2021 ).…”

“…Using a dual RNA-guided CRISPR endonuclease, such a Streptococcus pyogenes Cas9 (spCas9), prokaryotic organisms can specifically recognize and cleave invading foreign DNA ( Jiang and Doudna, 2017 ; Thompson et al, 2021 ). Crucially, the ability of Cas proteins to target, bind, and cleave selected nucleic acid sequences has been exploited for precise genome editing of eukaryotic organisms ( Jinek et al, 2012 ; Jiang and Doudna, 2017 ; Thompson et al, 2021 ). Hence, the development of CRISPR/Cas9 allowed for the rapid expansion of genome engineering into basic research ( Giersch and Finnigan, 2017 ; Thompson et al, 2021 ) as well as industrial biotechnology and synthetic biology ( Stovicek et al, 2015 ; Raschmanová et al, 2018 ; Mitsui et al, 2019 ; Zhang S. et al, 2020 ; Ding et al, 2020 ; Malcı et al, 2020 ; Meng et al, 2020 ; Molina-Espeja, 2020 ; Parapouli et al, 2020 ; Rainha et al, 2020 ; Patra et al, 2021 ).…”

“…Crucially, the ability of Cas proteins to target, bind, and cleave selected nucleic acid sequences has been exploited for precise genome editing of eukaryotic organisms ( Jinek et al, 2012 ; Jiang and Doudna, 2017 ; Thompson et al, 2021 ). Hence, the development of CRISPR/Cas9 allowed for the rapid expansion of genome engineering into basic research ( Giersch and Finnigan, 2017 ; Thompson et al, 2021 ) as well as industrial biotechnology and synthetic biology ( Stovicek et al, 2015 ; Raschmanová et al, 2018 ; Mitsui et al, 2019 ; Zhang S. et al, 2020 ; Ding et al, 2020 ; Malcı et al, 2020 ; Meng et al, 2020 ; Molina-Espeja, 2020 ; Parapouli et al, 2020 ; Rainha et al, 2020 ; Patra et al, 2021 ). Some important applications of CRISPR/Cas9 genome editing applications in S. cerevisiae involve the production of biopharmaceuticals, biocatalysts, food additives, chemicals, and biofuels ( Hong and Nielsen, 2012 ; Mattanovich et al, 2014 ; Auxillos et al, 2019 ; Mitsui et al, 2019 ; Kim et al, 2020 ; Lacerda et al, 2020 ; Molina-Espeja, 2020 ; Parapouli et al, 2020 ; Utomo et al, 2021 ).…”

“…Some important applications of CRISPR/Cas9 genome editing applications in S. cerevisiae involve the production of biopharmaceuticals, biocatalysts, food additives, chemicals, and biofuels ( Hong and Nielsen, 2012 ; Mattanovich et al, 2014 ; Auxillos et al, 2019 ; Mitsui et al, 2019 ; Kim et al, 2020 ; Lacerda et al, 2020 ; Molina-Espeja, 2020 ; Parapouli et al, 2020 ; Utomo et al, 2021 ). Moreover, the expanding CRISPR/Cas toolkit, which includes base editing ( Eid et al, 2018 ; Rees and Liu, 2018 ; Yang et al, 2019 ; Anzalone et al, 2020 ), gene repression and activation ( Gilbert et al, 2013 ; Qi et al, 2013 ; Didovyk et al, 2016 ; Dominguez et al, 2016 ; Brezgin et al, 2019 ; Pickar-Oliver and Gersbach, 2019 ; Xu and Qi, 2019 ; Shakirova et al, 2020 ) as well as alternative Cas proteins and Cas9 variants ( Bao et al, 2015 ; Nakade et al, 2017 ; Yan et al, 2019 ; Paul and Montoya, 2020 ; Thompson et al, 2021 ) have begun to greatly expand what can be accomplished with CRISPR/Cas systems in eukaryotic fungi. Our review broadly focuses on the application of various CRISPR technologies for manipulating the genome of Saccharomyces cerevisiae .…”