Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells

Zhang, Yilan; Ge, Xianglian; Yang, Fayu; Zhang, Liping; Zheng, Jiayong; Tan, Xiao; Jin, Zi‐Bing; Qu, Jia; Gu, Feng

doi:10.1038/srep05405

Cited by 186 publications

(178 citation statements)

References 19 publications

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“…The extent to which dCas9 can recognize PAM sequences aside from GG dinucleotides-known as noncanonical PAMs-has been the subject of conflicting reports (7,18,19). At 10 nM dCas9, the initial association rate of NGA or NAG targets was similar to the PAM-scanning behavior we described; however, after equilibration 12 h later, both NGA and NAG PAMs exhibited more signal than other PAM mutants (Fig.…”

Section: −280mentioning

confidence: 66%

High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

Boyle

Andreasson

Chircus

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

169

193

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The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nucleasedead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.DNA | molecular biophysics | kinetics | sequencing | CRISPR C RISPR-associated protein 9 (Cas9) is programmed to bind its target DNA by a guide RNA (gRNA) that, once loaded, forms a ribonucleoprotein (RNP) complex. The Streptococcus pyogenes CRISPR system, the most extensively studied and applied system to date, targets a 23-bp DNA sequence containing (i) an "NGG" protospacer adjacent motif (PAM) element downstream of the single-guide RNA (sgRNA) target DNA (1) and (ii) a 20-bp sequence upstream of the PAM bearing complementarity to the gRNA (2). Genome engineering applications leverage the nuclease activity of the Cas9 RNP, but Cas9 engineered to lack the residues required for cleavage [dCas9 (nuclease-dead Cas9)] has proven valuable by enabling the creation of customizable and programmable DNA binding elements that can activate and repress gene expression with high precision (CRISPRa and CRISPRi) (3).The biophysical underpinnings of the Cas9 target search have been investigated both by directed biochemical assays (4, 5) and through measurements of off-target Cas9 activity (6-11). These studies have led to a model for binding wherein Cas9 proceeds through a series of steps starting with PAM recognition, followed by DNA melting, RNA strand invasion, and heteroduplex formation dependent on complementarity with a 5-10-bp seed. Structural data have further suggested that conformational changes in the HNH domain reposition catalytic residues and permit allosteric regulation of the RuvC domain. This conformational gating ensures that cleavage occurs only in the context of substantial homology between gRNA and target (12, 13).The specificity of Cas9 DNA binding is crucial for all potential applications of Cas9's RNA-programmable targeting. Localization of dCas9...

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Section: −280mentioning

confidence: 66%

High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

Boyle

Andreasson

Chircus

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

169

193

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“…Wild-type SpCas9 has been shown to have some activity on sites with NAG and NGA PAMs, but typically with much lower efficiencies than on sites with canonical NGG PAMs Hsu et al, 2013;Zhang et al, 2014;Kleinstiver et al, 2015b). A recent study used a bacterial selection system to identify three new variants of SpCas9 that can target NGA, NGAG, and NGCG PAMs with high efficiencies and specificities (Table 1) (Kleinstiver et al, 2015b).…”

Section: Expanding the Targeting Scope Of Cas9mentioning

confidence: 99%

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

Komor¹,

Badran²,

Liu³

2017

Cell

259

204

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The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this review we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and some of the remarkable advancements in basic research, biotechnology, and therapeutics development that these developments have facilitated.

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“…NGG trinucleotide is a prerequisite for a guide RNA to drive Cas9 nuclease to cleave the specific portion of DNA. However noncanonical PAMs such as NAN, NTG and have also been recognized that can potentially drive nuclease to cleave the off target portion of genome thus leading to off target mutations 60 . Cas9 has been found to bind to a sequence with as many as 10 mismatches in the spacer of crRNAs 61 , yet reported to cleave and induce mutations at sequences with 3-5 mismatches 62 .…”

Section: Crispr Cas Challengesmentioning

confidence: 99%

Optimizing Crispr Cas9 Genome Editing System:A Review

Budhthoki¹,

Sharma²,

Khan³

et al. 2017

Int. j. endorsing health sci. res.

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CRISPR Cas9 is highly advanced genome editing technology extensively used for the modifications of genetic components in various sectors of living organisms. This technology has been adapted from the prokaryotic immune system, where it plays a vital role in protecting bacteria and archaea from virus attacks. This robust technology has currently been proven efficient in selective and precise editing, the genome of different living organisms for different purposes ranging from therapeutic, diagnostic to programmable gene regulation. This technology has been continuously upgraded, enhancing its performance thus reducing unfavorable outcomes. Customizing this technology is not a piece of cake. Hundreds of thousands of experiments have been conducted all around the world to optimize this highly intellectual technology to make it an error prone programmable technology to serve each and every living kind. In this review, we have summarized the modifications that have been made in different components of CRISPR cas9 system, engineering of CRISPR Cas9 for specific purposes, different external factors that has to be considered to obtain the best possible outcome minimizing the hazards.

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Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells

Cited by 186 publications

References 19 publications

High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

Optimizing Crispr Cas9 Genome Editing System:A Review

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