A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells

Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Ding, Chen; Hao, Xi Shan; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia; Gu, Feng

doi:10.1093/nar/gkx783

Cited by 104 publications

(139 citation statements)

References 22 publications

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“…PAM profiling of FnCas12a variants is shown in Figure S1 and S2. As expected, the efficiency of FnCas12a‐mediated DNA cleavage of TTV, TCV, CTV (V = A, C or G) PAMs was much higher than that of other recognizable PAMs like TTT and TCT, supporting the previously reported findings (L. Gao et al, ; Tu et al, ). Compared with WT FnCas12a, the FnCas12a variant N607R/K613V/N617R (hereafter referred to EP2), which is the analog to AsCas12a variant RVR, showed decreased activity and decreased PAM recognition sites.…”

Section: Resultssupporting

confidence: 90%

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Wang

Liu

et al. 2019

Biotech & Bioengineering

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As the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a (previously known as Cpf1) system cleaves double-stranded DNA and produces a sticky end, it could serve as a useful tool for DNA assembly/editing. To broaden its application, a variety of engineered FnCas12a proteins are generated with expanded protospacer adjacent motif (PAM) requirements. Two variants (FnCas12a-EP15 and EP16) increased the targeting range of FnCas12a by approximately fourfold. They can efficiently recognize a broad range of PAM sequences including YN (Y = C or T), TAC and CAA. Meanwhile, based on our demonstration that FnCas12a is active from 16 to 60°C, we developed an "improved CRISPR-Cas12a-assisted one-pot DNA editing" (iCOPE) method to facilitate DNA editing by combining the crRNA transcription, digestion, and ligation in one pot. By applying iCOPE, the editing efficiency reached 72-100% for two DNA fragment assemblies, and for the 21 kb large DNA construct modification, the editing efficiency can reach 100%. Thanks to the advantages of Cas12a, iCOPE with only one digestion enzyme could replace current a variety of restriction enzymes to perform the cloning in one pot with almost no sequence constraints. Taken together, this study offers an expanded DNA targeting scope of CRISPR systems and could serve as an efficient seamless one-pot DNA editing tool. K E Y W O R D SCas12a variants, DNA editing, FnCas12a, one pot, synthetic biology

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Section: Resultssupporting

confidence: 90%

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Wang

Liu

et al. 2019

Biotech & Bioengineering

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“…In contrast, the reducing reagents DTT and β‐ME were able to reduce the aggregated form of SaCas9 and allowed its reactivation (Figure B). This phenomenon was also observed during the purification of FnCpf1 (unpublished data) . In addition, SpCas9 purified by nickel affinity chromatography also showed some aggregates but smaller than the mono‐SpCas9 .…”

Section: Resultssupporting

confidence: 61%

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes

Wang

Xie

et al. 2019

Biotechnology Journal

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Genome editing using RNA‐guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5′‐NNGRRT‐3′, compared with Streptococcus pyogenes Cas9 (SpCas9) of 5′‐NGG‐3′ PAM. Here, SaCas9‐RNP‐mediated genome editing is reported in human cells. The SaCas9‐RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9‐RNP is successfully implemented to correct a pathogenic RS1 mutation for X‐linked juvenile retinoschisis. It is also shown that off‐target effects triggered by SaCas9‐RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9‐RNP‐mediated genome editing in human cells, which could facilitate genome‐editing‐based therapy.

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“…1B) [64,65]. However, more recent experiments revealed that FnCas12a possesses robust DNA cleavage activity in human cells as well with frequencies comparable to those of the other orthologues [67]. Instead of G-rich PAMs required by Cas9, Cas12a recognizes T-rich PAMs and thus further increases the number of potential target sites.…”

Section: Using Cas12a (Formerly Named Cpf1) In Plantsmentioning

confidence: 99%

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

2018

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Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way.

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A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells

Cited by 104 publications

References 22 publications

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

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