Abstract
Although most of the efforts to enhance the specificity and efficiency of CRISPR genome editing have focused on the Cas effectors and guide RNA selection, an increasing body of literature is making a case for engineering the guides themselves. Given the current enthusiasm about RNA technologies and increasing interest in the deployment of DNA-free ribonucleotide protein (RNP) complexes to perform genome editing, further enhancing the guide RNA is a sensible approach. After all, it is technically the only remnant of a bona fide clustered regularly interspaced short palindromic repeat (a piece of the repeat for commonly used Cas9 guides) in CRISPR technology.
Early efforts on guide sequence, length, and structure have been supplanted recently by chemistry-informed inclusion of modified nucleosides to promote guide stability and interactions with the Cas effector as well as the target nucleic acid to form more specific and discriminative molecular interactions. In this issue of The CRISPR Journal, multiple groups document the engineering of CRISPR RNA guides with various designs and nucleosides to modulate activity.
To enhance CRISPR interference screens, Mills et al. (see page 769) present chemically modified synthetic single-guide RNAs in combination with a dCas9 fusion with a Sal-like protein 1 domain (SALL1) and a Sin3a corepressor complex component (SDS3). The authors show that this dCas9-SALL1-SDS3 fusion protein can be transcribed in vitro into mRNA that can be co-delivered with synthetic sgRNAs (encompassing 2′-O-methyl phosphorothioate modifications at both termini) for potent and specific transcriptional repression in clinically important cell types (e.g., human-induced pluripotent stem cells and primary human T cells).
Given the technical difficulties and financial inefficiencies inherent to the synthesis of RNA molecules longer than 100 nucleotides, Hoy et al. (see page 787) present a bio-orthogonal chemistry-based conjugation approach that enables the relatively efficient assembly of two shorter nucleotides: a tetrazine-containing 31-mer with a trans-cylcooctene-modified 70mer. Inspired by previous study on “split and click” chemistry, the authors show that such sgRNA libraries can be synthesized to encompass diverse chemical modifications (m1A, m6A, s2U, and S4U) with functional promise in HEK293T human cells.
Besides designing and engineering affordable guides that promote stability, efficiency, and specificity, researchers also need to account for the tolerability of genome editing molecular machines. Prokhorova et al. (see page 799) address tolerability by modifying guide RNAs to reduce cytotoxicity and immunogenicity. The authors integrated N6-methyladenosine (m6A), 5-methylcitidine (m5C), and pseudouridine into guide RNAs and tracrRNAs.
As CRISPR-based technologies are increasingly tested in the clinic, such efforts in improving guide RNAs illustrate the continued need to optimize the activity and specificity of genome editing, while limiting potential cytotoxic and immunogenic effects, and ensuring practical scale-up and affordability. Besides better guides per se, it is important for the community to also provide guidance to regulatory agencies as those technologies get deployed outside of laboratory contexts.
As featured in other articles in this year-end issue of The CRISPR Journal, our community continues to advance CRISPR technologies and their uses. We are pleased to publish a timely review on prime editing in mammals, as well as research studies on genome editing in mosquitos and large deletions in pigs. These studies reflect the broad potential of these technologies across the tree of life and the continued diversification of the applications of CRISPR. Collectively, these advances are also important to document safety and provide confidence in enhanced efficacy to regulators, patients and stakeholders involved in the clinical and translational aspects of human genome editing.
Five in the Hand
Finally, it is a pleasure to note that this issue marks the conclusion of volume 5 of The CRISPR Journal. It is hard to believe we launched the journal 5 years ago. The years have flown by, even if there were moments for the past few years when time seemed to stand still. We thank everyone who has contributed to the journal's success—authors, referees, editorial board members, subscribers, and readers. We are excited for what the field of CRISPR and genome editing will reveal in the years ahead. Look for some important announcements early in the New Year.