Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a 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 off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a 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–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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

10.1101/2020.02.04.933614 ◽

2020 ◽

Author(s):

Hanseop Kim ◽

Wi-jae Lee ◽

Seung-Hun Kang ◽

Junho K. Hur ◽

Hyomin Lee ◽

...

Keyword(s):

Dna Sequences ◽

Genome Engineering ◽

Target Genes ◽

Genetic Mutation ◽

Target Sequence ◽

Energy Potential ◽

Guide Rna ◽

Sequence Recognition ◽

Effective Manner ◽

Target Dna

AbstractThe 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 off-target 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 off-target 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 diseases caused by genetic mutation.

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A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

Science Advances ◽

10.1126/sciadv.1600699 ◽

2016 ◽

Vol 2 (8) ◽

pp. e1600699 ◽

Cited By ~ 11

Author(s):

Hiroshi Arakawa

Keyword(s):

Dna Sequences ◽

Genetic Screening ◽

Restriction Enzymes ◽

Mrna Sequence ◽

Complementary Sequence ◽

Complementary Dna ◽

Random Primer ◽

Guide Rna ◽

Protospacer Adjacent Motif ◽

Target Dna

The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.

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Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012239 ◽

2020 ◽

Vol 295 (19) ◽

pp. 6509-6517 ◽

Cited By ~ 1

Author(s):

Vladimir Mekler ◽

Konstantin Kuznedelov ◽

Konstantin Severinov

Keyword(s):

Genome Editing ◽

Target Location ◽

Dna Probes ◽

Target Sequence ◽

Francisella Novicida ◽

Guide Rna ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Protospacer Adjacent Motif ◽

Target Dna

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

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In vitro analysis of site specific nuclease selectivity by NGS

AIMS Bioengineering ◽

10.3934/bioeng.2021020 ◽

2021 ◽

Vol 8 (4) ◽

pp. 235-242

Author(s):

Vincent Brondani ◽

Keyword(s):

Restriction Endonuclease ◽

Genome Engineering ◽

Direct Interaction ◽

Target Sequence ◽

Vitro Assay ◽

Guide Rna ◽

Phosphate Backbone ◽

Target Dna ◽

Vitro Analysis

<abstract> <p>Nucleases currently used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. The RNA guided nucleases describe today are recognizing a sequence with two distinct molecular interactions: first, like a restriction endonuclease, by direct interaction between the protein and the DNA; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report an in vitro assay to assess the cleavage specificity and the selectivity of the nucleases. The assay is designed using a plasmid encompassing the DNA target site degenerated at positions determined on structural feature. The results demonstrate that the Cpf1 RNA guided nuclease is highly specific for the target sequence, nevertheless its substrate selectivity is low compare to a restriction endonuclease.</p> </abstract>

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Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

10.1101/600890 ◽

2019 ◽

Cited By ~ 8

Author(s):

Ryan T. Fuchs ◽

Jennifer Curcuru ◽

Megumu Mabuchi ◽

Paul Yourik ◽

G. Brett Robb

Keyword(s):

Dna Sequences ◽

Nuclease Activity ◽

Target Sequence ◽

Sequence Composition ◽

Guide Rna ◽

Dna Targeting ◽

Single Turnover ◽

Target Dna ◽

Buffer Composition

ABSTRACTCRISPR-Cas12a (Cpf1) are RNA-guided nuclease effectors of acquired immune response that act in their native organisms by cleaving targeted DNA sequences. Like CRISPR-Cas9 RNA-guided DNA targeting enzymes, Cas12a orthologs have been repurposed for genome editing in non-native organisms and for DNA manipulationin vitro. Recent studies have shown that activation of Cas12a via guide RNA-target DNA pairing causes multiple turnover, non-specific ssDNA degradation intrans, after single turnover on-target cleavage incis. We find that the non-specifictransnuclease activity affects RNA and dsDNA in addition to ssDNA, an activity made more evident by adjustment of reaction buffer composition. The magnitude of thetransnuclease activity varies depending on features of the guide RNA being used, specifically target sequence composition and length. We also find that the magnitude oftransnuclease activity varies between the three most well-studied Cas12a orthologs and that the Cas12a fromLachnospiraceaebacterium ND2006 appears to be the most active.

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CRISPR-Cas9 bends and twists DNA to read its sequence

10.1101/2021.09.06.459219 ◽

2021 ◽

Cited By ~ 2

Author(s):

Joshua C. Cofsky ◽

Katarzyna M. Soczek ◽

Gavin J. Knott ◽

Eva Nogales ◽

Jennifer A. Doudna

Keyword(s):

Genome Editing ◽

Target Sequence ◽

Base Flipping ◽

Base Pairs ◽

Guide Rna ◽

Target Capture ◽

Dna Base ◽

Protospacer Adjacent Motif ◽

Target Dna ◽

Dna Base Pairs

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM)1. Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism2,3. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

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Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1619926114 ◽

2017 ◽

Vol 114 (21) ◽

pp. 5443-5448 ◽

Cited By ~ 30

Author(s):

Vladimir Mekler ◽

Leonid Minakhin ◽

Konstantin Severinov

Keyword(s):

Dna Sequences ◽

Rna Binding ◽

Proximal Region ◽

Target Sequence ◽

Duplex Dna ◽

Rna Molecules ◽

Guide Rna ◽

Double Stranded Dna ◽

Protospacer Adjacent Motif ◽

Dna Complex

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

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CRISPR-Cas9 bends and twists DNA to read its sequence

10.21203/rs.3.rs-882403/v1 ◽

2021 ◽

Author(s):

Jennifer Doudna ◽

Joshua Cofsky ◽

Katarzyna Soczek ◽

Gavin Knott ◽

Eva Nogales

Keyword(s):

Genome Editing ◽

Target Sequence ◽

Base Flipping ◽

Base Pairs ◽

Guide Rna ◽

Target Capture ◽

Dna Base ◽

Protospacer Adjacent Motif ◽

Target Dna ◽

Dna Base Pairs

Abstract In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

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Developing Heritable Mutations in Arabidopsis thaliana Using a Modified CRISPR/Cas9 Toolkit Comprising PAM-Altered Cas9 Variants and gRNAs

Plant and Cell Physiology ◽

10.1093/pcp/pcz118 ◽

2019 ◽

Vol 60 (10) ◽

pp. 2255-2262 ◽

Cited By ~ 5

Author(s):

Akihiro Yamamoto ◽

Takashi Ishida ◽

Mika Yoshimura ◽

Yuri Kimura ◽

Shinichiro Sawa

Keyword(s):

Arabidopsis Thaliana ◽

Genome Editing ◽

Target Genes ◽

Molecular Genetic ◽

Science Research ◽

Target Sequence ◽

Guide Rna ◽

Cas9 Nuclease ◽

Protospacer Adjacent Motif ◽

Model Plant Arabidopsis Thaliana

Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), comprising an RNA-guided DNA endonuclease and a programmable guide RNA (gRNA), is currently recognized to be a powerful genome-editing tool and is widely used in biological science. Despite the usefulness of the system, a protospacer-adjacent motif (PAM) immediately downstream of the target sequence needs to be taken into account in the design of the gRNA, a requirement which limits the flexibility of the CRISPR-based genome-editing system. To overcome this limitation, a Cas9 isolated from Streptococcus pyogenes, namely SpCas9, engineered to develop several variants of Cas9 nuclease, has been generated. SpCas9 recognizes the NGG sequence as the PAM, whereas its variants are capable of interacting with different PAMs. Despite the potential advantage of the Cas9 variants, their functionalities have not previously been tested in the widely used model plant, Arabidopsis thaliana. Here, we developed a plant-specific vector series harboring SpCas9-VQR (NGAN or NGNG) or SpCas9-EQR (NGAG) and evaluated their functionalities. These modified Cas9 nucleases efficiently introduced mutations into the CLV3 and AS1 target genes using gRNAs that were compatible with atypical PAMs. Furthermore, the generated mutations were passed on to their offspring. This study illustrated the usefulness of the SpCas9 variants because the ability to generate heritable mutations will be of great benefit in molecular genetic analyses. A greater number of potential SpCas9-variant-recognition sites in these genes are predicted, compared with those of conventional SpCas9. These results demonstrated the usefulness of the SpCas9 variants for genome editing in the field of plant science research.

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