Improving FnCas12a Genome Editing by Exonuclease Fusion

Wu, Yongqiang; Yuan, Qichen; Zhu, Yu; Gao, Xiang; Song, Jiabao; Yin, Ziru

doi:10.1089/crispr.2020.0073

Cited by 15 publications

(6 citation statements)

References 32 publications

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“…To this end, we first created fusion proteins in which the carboxy-terminus of SpyCas9 was linked with a DNA-modifying protein-either a 5′ to 3′ DNA exonuclease (RecE, RecJ, T5, or lambda) 24 , mung bean nuclease, or terminal deoxynucleotidyl transferase (TdT). T5 and RecJ exonucleases were reported to enhance FnCas12a, T5-FnCas12a increased the knockout efficiency of FnCas12a at multiple genomic loci in different human cell lines, while RecJ-FnCas12a decreased the knockout efficiency of FnCas12a 17 .…”

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confidence: 96%

“…In addition, a SpyCas9 chimeric fusion protein with the three prime repair exonuclease 2 (TREX2), a human 3′ to 5′ exonuclease involved in DNA repair, replication, and recombination, was also reported to increase mutagenic efficiency 15,16 . TEXT (Tethering EXonuclease T5 with FnCas12a)-a fusion strategy significantly increased the knockout efficiency of FnCas12a at multiple genomic loci in different human cell lines 17 . In case of the knock-in activity, when the human CtIP endonuclease was fused to SpyCas9, the chimeric protein resulted in more than two-fold increase in transgene integration efficiency relative to SpyCas9 backbone protein alone 18 .…”

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confidence: 99%

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Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

Yoon¹,

Kwon²,

Han³

et al. 2021

Sci Rep

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Efforts to improve CRISPR-Cas9 genome editing systems for lower off-target effects are mostly at the cost of its robust on-target efficiency. To enhance both accuracy and efficiency, we created chimeric SpyCas9 proteins fused with the 5′-to-3′ exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600% without noticeable increase in off-target effects. Improved activity of the two fusion proteins should enable editing of the previously hard-to-edit genes and thus readily obtaining the cells with designer traits.

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confidence: 96%

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confidence: 99%

Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

Yoon¹,

Kwon²,

Han³

et al. 2021

Sci Rep

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“…It would be ideal to further modify ErCas12a as a heatshock-free tool for convenient and embryo-friendly applications. Previous reports suggested that co-transfection of DNA exonuclease with Cas9 in human induced pluripotent stem cells increased the mutagenesis rate [35], while the fusion of the DNA exonuclease to the N-terminus or C-terminus of FnCas12a improved its gene editing efficiency to a similar or even higher level compared to those of LbCas12a and AsCas12a [36]. Thus, we first employed the commonly used human 3' exonuclease TREX2 and fused it to either the N-or C-terminus of ErCas12a and tested its efficiency using tyr pre-crRNA and alb pre-crRNA.…”

Section: T5exo-ercas12a Efficiently Induces Indel Mutation and Promot...mentioning

confidence: 97%

ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

Han

Zhang

Yang

et al. 2022

Biology

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In zebrafish, RNA-guided endonucleases such as Cas9 have enabled straightforward gene knockout and the construction of reporter lines or conditional alleles via targeted knockin strategies. However, the performance of another commonly used CRISPR system, Cas12a, is significantly limited due to both the requirement of delivery as purified protein and the necessity of heatshock of injected embryos. To explore the potential of CRISPR/Cas12a-mediated genome editing and simplify its application in zebrafish, we took advantage of the recently reported mRNA-active ErCas12a and investigated its efficacy for the knockin of large DNA fragments, such as fluorescent reporter genes. For knockin via either microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ) pathways, ErCas12a-injected embryos with a brief heatshock displayed comparable knockin efficiency with Cas9 injection. Through the fusion of T5 exonuclease (T5exo) to the N-terminus of ErCas12a (T5exo-ErCas12a), we further demonstrated high efficiency gene knockout and knockin at a normal incubation temperature, eliminating the embryo-damaging heatshock step. In summary, our results demonstrate the feasibility of ErCas12a- and T5exo-ErCas12a-mediated genome manipulation under simplified conditions, and further expand the genome editing toolbox for various applications in zebrafish.

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“…Cas Φ can be used for genome editing and DNA detection with only half the molecular mass of Cas9 or Cas12a genome editing enzymes. This facilitates its transformation, thus expanding genome editing tools [108,[122][123][124][125][126][127].…”

Section: Crispr-cas φmentioning

confidence: 99%

Research progress in gene editing technology

Huang

Yao

et al. 2021

Front. Biosci. (Landmark Ed)

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As a tool for modifying the genome, gene editing technology has developed rapidly in recent years, especially in the past two years. With the emergence of new gene editing technologies, such as transposon editing tools, numerous advancements have been made including precise editing of the genome, double base editing, and pilot editing. This report focuses on the development of gene editing tools in recent years, elaborates the progress made in classic editing tools, base editor and other new editing tools, and provides insights into challenges and opportunities.

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Improving FnCas12a Genome Editing by Exonuclease Fusion

Cited by 15 publications

References 32 publications

Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

Research progress in gene editing technology

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