Engineered FnCas12a with enhanced activity through directional evolution in human cells

Liu, Xiexie; Liu, Xiaoyu; Zhou, Chenchen; Lv, Ji‐Neng; He, Xiubin; Liu, Yuanyuan; Xie, Haihua; Wang, Bang; Lv, Xiujuan; Tang, Lianchao; Li, Mingchun; Liu, Changbao; Zhao, Junzhao; Liu, Yong; Song, Zongming; Gu, Feng

doi:10.1016/j.jbc.2021.100394

Cited by 16 publications

(9 citation statements)

References 39 publications

(51 reference statements)

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“…To overcome this challenge, both structure-guided and directed mutagenesis approaches have been applied to relax the PAM constraints of Cas12a, yielding variants with broadened ranges. Furthermore, several Cas12a variants with increased efficiency have been engineered. − Notably, mutations that accommodate noncanonical PAMs also improve cleavage at canonical sites, and hyperactivating mutations can substantially enhance Cas12a activity across a wide range of primary cell types …”

Section: Harnessing Class 2 Crispr Diversity For Applications In Geno...mentioning

confidence: 99%

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

2023

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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.

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Section: Harnessing Class 2 Crispr Diversity For Applications In Geno...mentioning

confidence: 99%

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

2023

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“…Cas12a nucleases display an A/T-rich PAM, promoting effective base editing in sites where Cas9 cannot target . Analogous to Cas9, Cas12a possesses diverse orthologs contributing to base editing, such as orthologs from Francisella novicida (FnCas12a), Lachnospiraceae bacterium (LbCas12a), and Acidaminococcus sp. (AsCas12a) . Apart from different PAM preference, Cas12a offers unique advantages over Cas9, such as shorter crRNA requirement, low mismatch tolerance, low levels of unintended indel generation, and facile operations for multiplex editing. , Recently, the Cas12f orthologs also have been employed for BEs.…”

Section: Optimization Strategies Based On Core Component Engineering ...mentioning

confidence: 99%

“…Cas12a nucleases display an A/T-rich PAM, promoting effective base editing in sites where Cas9 cannot target. 15 Analogous to Cas9, Cas12a possesses diverse orthologs contributing to base editing, such as orthologs from Francisella novicida (FnCas12a), 16 Lachnospiraceae bacterium (LbCa-s12a), 17 and Acidaminococcus sp. (AsCas12a).…”

Section: Expanding Pam Compatibility By Harnessing Casmentioning

confidence: 99%

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

Qin

Zhang

et al. 2023

ACS Synth. Biol.

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Base editors (BE) based on CRISPR systems are practical gene-editing tools which continue to drive frontier advances of life sciences. BEs are able to efficiently induce point mutations at target sites without double-stranded DNA cleavage. Hence, they are widely employed in the fields of microbial genome engineering. As applications of BEs continue to expand, the demands for base-editing efficiency, fidelity, and versatility are also on the rise. In recent years, a series of optimization strategies for BEs have been developed. By engineering the core components of BEs or adopting different assembly methods, the performance of BEs has been well optimized. Moreover, series of newly established BEs have significantly expanded the base-editing toolsets. In this Review, we will summarize the current efforts for BE optimization, introduce several novel BEs with versatility, and look forward to the broadened applications for industrial microorganisms.

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“…NG-ABEmax libraries were generated using the protocols which described previously 48 . For the library 1, the NG-ABEmax plasmids were digested with BseRI, and the digested products (backbone) was subsequently purified (8277 bp).…”

Section: Construction Of Ng-abemax Mutant Librariesmentioning

confidence: 99%

Human cells based directed evolution of adenine base editors with improved efficiency

Liu

et al. 2021

Preprint

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Adenine base editors (ABE) are novel genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion in genomic DNA. However, the low activity of ABE remains a major bottleneck that precludes efficacious applications. To address this limitation, we developed a directional screening system in human cells to evolve the deaminase component of the ABE, and identified three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we created a new, consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibited superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also found that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve novel, user-specified traits in base editors that extend beyond augmented editing activity.

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Engineered FnCas12a with enhanced activity through directional evolution in human cells

Cited by 16 publications

References 39 publications

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

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

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

Human cells based directed evolution of adenine base editors with improved efficiency

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