Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang

Moon, Su Bin; Lee, Jeongmi; Kang, Jeong Gu; Lee, Nan-Ee; Ha, Dae-In; Kim, Do Yon; Kim, Sun Hee; Yoo, Kwangsun; Kim, Daesik; Ko, Jeong–Heon; Kim, Yong-Sam

doi:10.1038/s41467-018-06129-w

Cited by 149 publications

(110 citation statements)

References 45 publications

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“…Cas12a has been successfully used for gene editing in vivo without any deleterious offtarget effects (D. Kim et al 2016;Kleinstiver et al 2016; H. K. Kim et al 2017;Tang et al 2018;Moon et al 2018). Our high-throughput pLibrary cleavage analysis indicates that Cas12a can bind and cleave sequences with up to four mismatches in vitro.…”

Section: Discussionmentioning

confidence: 86%

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Murugan

Seetharam

Severin

et al. 2019

Preprint

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Cas12a (formerly Cpf1) is an RNA-guided endonuclease in the CRISPR-Cas immune system that can be easily programmed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for precise genome editing applications.This specificity is contradictory to the natural role of Cas12a as an immune effector against rapidly evolving phages. However, the native cleavage specificity and activity remains to be fully understood. We employed high-throughput in vitro cleavage assays to determine and compare the native specificities of three Cas12a orthologs. Surprisingly, we observed pervasive nicking of randomized target libraries, with strong nicking activity observed against targets with up to four mismatches. Nicking and cleavage activities are dependent on mismatch type and position, and vary depending on the Cas12a ortholog and crRNA sequence. Our high-throughput and biochemical analysis further reveal that Cas12a has robust activated non-specific nicking and weak non-specific dsDNA degradation activity in trans. Together, our findings reveal Cas12a

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Section: Discussionmentioning

confidence: 86%

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Murugan

Seetharam

Severin

et al. 2019

Preprint

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“…Though FnCpf1 uses a more common TTN PAM sequence, its cleavage activity is still modest (Tu et al 2017). Editing efficiency of the Cas12a systems has since been enhanced by artificially engineering the crRNA to increase activity without a loss of specificity (Bin Moon et al 2018). Therefore, it is of interest to test various modifications of crRNAs and note their effect on mutagenesis and gene targeting going forward.…”

Section: Discussionmentioning

confidence: 99%

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

Wierson

Simone

WareJoncas

et al. 2019

Preprint

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) effector proteins enable the direction of DNA double-strand breaks at defined loci based on a variable length RNA guide specific to each effector.The guides are generally similar in size and form, consisting of a ~20 base sequence homologous to the DNA target and a secondary structure used by the effector for guide/nuclease recognition. However, the effector proteins vary in size, DNA binding kinetics, nucleic acid hydrolyzing activities, and Protospacer Adjacent Motif (PAM) requirements. Recently, a Cas12a family member protein named Mad7 was identified that is most similar to the Cas12a from Acidaminococcus sp. Here, we report for the first time Mad7 activity in zebrafish and human cells. We utilize a fluorescent reporter system to demonstrate CRISPR/Mad7 elicits strand annealing mediated DNA repair more efficiently than CRISPR/Cas9. Finally, we use CRISPR/Mad7 with our previously reported gene targeting method GeneWeld in order to integrate reporter alleles in both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR/Cas system, increasing the flexibility researchers have in applying genome engineering technologies.

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“…As a result, there was a significant increase in cleavage specificity (0.11~4.15 fold) for chimeric guides ((cr)RNA No. 14,20,21,22,23) that did not induce DNA off-target cleavage ( Figure 4B). For the DNMT1 target gene, consecutive DNA substitutions from the 3′-end of the guide to the eighth nucleotide resulted in less on-target compensation and higher target cleavage specificity than DNA substitutions in the seed region close to the PAM sequence ( Figures 4A, 4B).…”

Section: Target Dna Cleavage Specificity Of Chimeric Dna-rna-guided Cpf1mentioning

confidence: 97%

Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

Kim

Lee

Kang

et al. 2020

Preprint

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The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and offtarget cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less offtarget mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable 2 diseases caused by genetic mutation.

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Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang

Cited by 149 publications

References 45 publications

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

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