An appealing alternative to the Streptococcus pyogenes CRISPR nuclease SpyCas9, are Type V CRISPR Cas12a nucleases, commonly isolated from Acidaminococcus (Asp) and Lachnospiraceae (Lba). These Cas12a nucleases embody several desirable attributes that SpyCas9 lacks: they exhibit greater editing precision, recognize a thymine-rich PAM (protospacer adjacent motif—a two-to-six base sequence following the nuclease target), use a single CRISPR-RNA to detect its target, cut DNA in a staggered fashion generating overhangs, process CRISPR arrays, and have been shown to function in diverse organisms ranging from plants to mammals. However, Cas12a nucleases exhibit lower editing rates than SpyCas9 in primary cells.

Scot Wolfe, PhD, professor of molecular, cell and cancer biology at the University of Massachusetts Chan Medical School, is the senior author of this study.

In a study published in GEN magazine’s sister journal, GEN Biotechnology (“Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells”), Scot Wolfe, PhD, professor of molecular, cell and cancer biology at the University of Massachusetts Chan Medical School and his team, increased Cas12a’s on-target gene editing rate to nearly 100% by engineering the configuration of the enzyme’s nuclear localization signal (NLS). These advancements to the Cas12a editing framework could improve the use of this nuclease to uncover functions of new genes and develop new CRISPR-based treatments.

“Previous work by our laboratories and others indicated that the efficiency of Cas12a editing in CD34+ hematopoietic stem and progenitor cells could potentially be improved by increasing the efficiency of its nuclear import,” said Wolfe.

In earlier studies, Wolfe’s team had enhanced SpyCas9 gene editing in primary cells by optimizing the NLS sequence composition and number. They had found adding one NLS at the amino-terminus and two at the carboxy-terminus of the nuclease markedly improved SpyCas9’s (3xNLS-SpyCas9) editing efficiency in hematopoietic stem and progenitor cells (HSPCs). They had then added two NLSs to the carboxy-terminus of Cas12a but did not achieve the same efficiency of targeted mutagenesis as the engineered SpyCas9 with three NLSs.

Ben Kleinstiver, PhD, assistant professor of pathology at Massachusetts General Hospital and Harvard Medical School, said, “Genome editing efficiency is impacted by many different variables, including the concentration of a CRISPR-Cas enzyme in the nucleus where it performs its function. Researchers have previously dedicated substantial effort to improve CRISPR nuclease expression and nuclear localization for SpyCas9, but comparatively fewer optimizations have been performed for Cas12a.” (Kleinstiver was not involved in the current study).

Kevin Luk, PhD, a former student in Wolfe’s lab and currently a scientist at Excision BioTherapeutics, is the first author of the study.

In the current study, Wolfe’s team developed three NLS C-terminus variants of Cas12a where they substituted the previously used simian virus NLS (SV40) with a more efficient NLS of a proto-oncogene (c-Myc). In addition, they added a third NLS to the carboxy end to achieve an editing platform at par with 3xNLS-SpyCas9 in editing efficiency. The researchers observed increased knockout efficiency in all three Cas12a orthologs (Asp, Lba, and engineered-Asp) they tested, which suggests this triple NLS strategy could be effective in improving the activity of other members of the Cas12a family, without decreasing the enzyme’s inherent specificity.

The study used standard electroporation to deliver the engineered Cas12a ribonucleoproteins (RNPs) into transformed human cells lines (HEK293T, Jurkat, and K562 cells) and into primary cells (natural killer cells and CD34+ HSPCs) to improve indel frequencies.

“We believe that the improved NLS sequence architecture described in this paper will increase the efficiency of genome editing by Cas12a in primary cells, thus leading to increased levels of therapeutic genome editing in a variety of applications,” said Wolfe. The researchers claim this strategy of enhancing the NLS sequence can be widely applied to other Cas12a orthologs and variants with similar outcomes.

“The Wolfe lab and collaborators had previously demonstrated increased activity with a new NLS framework for SpyCas, so it is exciting that they demonstrated success with a new NLS for Cas12a in this publication. It is important to have additional NLSs to test in the growing list of nucleases and cell types,” said Thomas Cradick, PhD, CSO at Excision BioTherapeutics. (Cradick was not involved in the current study.)

Kleinstiver said, “Luk et al., demonstrated that the efficiency of editing with various Cas12a enzymes can be improved by using a more optimal configuration of NLSs. The effect of this optimization was most striking in lipid-based transfections (nucleofections) in transformed cell lines, with a more modest improvement in primary cells, the latter of which due to already high levels of editing in primary cells.”

“This study resurfaces a really important consideration, that you can only edit cells as efficiently as your enzyme is designed to. There are lots of knobs to turn to optimize and improve editing efficiency, and the NLS architecture clearly plays a key role in regulating the nuclear concentration, and thus the potency, of the editor,” added Kleinstiver.

Nicole Gaudelli, PhD, director and head of gene editing platform technologies at Beam Therapeutics, who was not involved in the current study, said, “In addition to advancing Cas12a gene editing applications, these learnings may potentially be evaluated for other gene editing tools to further increase editing efficiencies and provide greater therapeutic benefit if higher levels of gene correction or modification can be achieved.”

“This study was rigorously done in multiple cell types that show the robustness of the data. I liked how they delivered Cas12 as an RNP, as this is therapeutically relevant and greatly reduces off-target editing,” said Alexis Komor, PhD, assistant professor of chemistry and biochemistry at the University of California, San Diego, who was not involved in the study.

“I also liked this work because it uses a very universal approach to improve editing (the modifications they made to the system can be applied to any genome editing agent), and they demonstrated its utility with multiple Cas12 enzymes (which have slightly different PAMs, which is nice). Overall, it’s a useful and practical study,” Komor continued.

“As we continue the deployment of diverse CRISPR-Cas effectors in the clinic, it is important to individually engineer each molecular machine for optimal efficiency and specificity. Here, the authors show how NLS can be optimized for enhanced activity in medically relevant human primary cells,” said Rodolphe Barrangou, PhD, professor of food, bioprocessing, and nutrition at North Carolina State University (NCSU), editor-in-chief of The CRISPR Journal, and CEO of TreeCo, a company that uses CRISPR to produce genetically enhanced trees. Barrangou was not part of the current study.

“Optimizing on-target mutagenesis rates whilst maintaining specificity is key for successful translation to the clinic,” reaffirmed Jennifer Harbottle, PhD, a senior scientist at Horizon Discovery, who was not part of this study. “The Cas12a NLS variant developed by Scot Wolfe’s lab holds the potential to lower dosage whilst exerting therapeutic effect.”

“It will be of interest to see this strategy expanded to other Type V systems, and track efficiency of delivery in a wider range of cell types and tissues,” added Harbottle. “Comprehensively evaluating the genomic integrity of edited cells, particularly the occurrence of structural variants and chromosomal rearrangements compared to editing by canonical Cas9 systems, will be critical to push the optimized Type V variants towards in vivo use in humans.”

In future studies, Wolfe intends to continue refining Cas12a nucleases to edit specific therapeutic targets. He said, “We are particularly interested in applications for certain hematopoietic disorders and muscular dystrophies.”

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