scholarly journals Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application

2021 ◽  
Vol 12 ◽  
Author(s):  
Qibing Liu ◽  
Siwei Wang ◽  
Juying Long ◽  
Zhuoyue Chen ◽  
Bing Yang ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Gene Editing ◽  
Xanthomonas Oryzae ◽  
Functional Study ◽  
Target Sequence ◽  
Type I ◽  
Functional Identification ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
New Vision

The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5′-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99A strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.

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

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

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.

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

2020 ◽  
Vol 295 (19) ◽  
pp. 6509-6517 ◽  
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.

scholarly journals Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2099
Author(s):  
Yunxing Liu ◽  
Fang Liang ◽  
Zijiong Dong ◽  
Song Li ◽  
Jianmin Ye ◽  
...  
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Gene Editing ◽  
Zebrafish Genome ◽  
Human Cells ◽  
Ribonucleoprotein Complex ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Low Activity

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

2021 ◽  
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.

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

2017 ◽  
Vol 114 (21) ◽  
pp. 5443-5448 ◽  
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.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

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.

scholarly journals Kinetic basis for DNA target specificity of CRISPR-Cas12a

2018 ◽  
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
James R. Rybarski ◽  
Ilya J. Finkelstein ◽  
Rick Russell
Keyword(s):  
Dna Cleavage ◽  
Target Sequence ◽  
Seed Region ◽  
Target Specificity ◽  
Loop Formation ◽  
Guide Rna ◽  
Dna Targeting ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Late Transition

SUMMARYClass II CRISPR-Cas nucleases are programmable via a single guide RNA, enabling genome editing applications in nearly all organisms. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically-separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite the functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence, implying substantial reversibility during R-loop formation –a late transition state– and the absence of a ‘seed’ region. Our results provide a quantitative underpinning for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity of Cas12a than the Cas9 nuclease.

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

2019 ◽  
Vol 60 (10) ◽  
pp. 2255-2262 ◽  
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.

scholarly journals CRISPR - CAS9 GENE EDITING: A REVIEW

2020 ◽  
Vol 8 (10) ◽  
pp. 1127-1132
Author(s):  
Devam Desai ◽  
◽  
Hiral Panchal ◽  
Shivani Patel ◽  
Ketul Nayak
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Target Sequence ◽  
Antiviral Defense ◽  
Defense System ◽  
Guide Rna ◽  
Therapeutic Applications ◽  
Current Review ◽  
Cas9 Nuclease ◽  
Cost Efficient

CRISPR is an RNA guided genome editing technique of genetic engineering which works like genetic scissors. Based on simplified version of bacterial CRISPR-Cas9 antiviral defense system. It is more accurate, faster and cost efficient than other genome editing methods. There are two components in this system: First component includes a single guide RNA (sgRNA) of system which will identify target sequence in genome and Second component will include Cas9 nuclease of system which will act as a pair of scissors to spilt the double strands of DNA. CRISPR has promising therapeutic applications. This current review focuses on mechanism, therapeutic applications, delivery systems, limitations and different approaches used for gene editing using CRISPR.

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