CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

/pdf/high-fidelity-crispr-cas9-nucleases-with-no-detectable-22u8sim672.pdf

High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects

CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics

/pdf/crispr-cas12a-target-binding-unleashes-indiscriminate-single-3wl0bk4y7k.pdf

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

https://science.sciencemag.org/content/sci/early/2015/11/30/science.aad5227.full.pdf

Rationally Engineered Cas9 Nucleases With Improved Specificity

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.

/pdf/nucleic-acid-detection-with-crispr-cas13a-c2c2-4enk3eyocr.pdf

Nucleic acid detection with CRISPR-Cas13a/C2c2

http://stacks.cdc.gov/view/cdc/30086/cdc_30086_DS1.pdf

Crystal structure of Cas9 in complex with guide RNA and target DNA

http://stacks.cdc.gov/view/cdc/37605/cdc_37605_DS1.pdf

Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

The RNA-guided endonuclease Cas9 has been harnessed as a tool for genome editing in mammalian cells1, 2. In addition, strategies employing catalytic inactive Cas9 can direct effector proteins to genomic targets3-5 to achieve transcriptional modulation. Here, we demonstrate that Cas9 can be split into two fragments and rendered chemically inducible by rapamycin sensitive dimerization domains for controlled reassembly to mediate genome editing and transcription modulation.

/pdf/a-split-cas9-architecture-for-inducible-genome-editing-and-27exdo7vx6.pdf

A Split Cas9 Architecture for Inducible Genome Editing and Transcription Modulation

The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System

Cas9-based technologies have transformed genome engineering and the interrogation of genomic functions, but methods to control such technologies across numerous dimensions-including dose, time, specificity, and mutually exclusive modulation of multiple genes-are still lacking. We conferred such multidimensional controls to diverse Cas9 systems by leveraging small-molecule-regulated protein degron domains. Application of our strategy to both Cas9-mediated genome editing and transcriptional activities opens new avenues for systematic genome interrogation.

/pdf/multidimensional-chemical-control-of-crispr-cas9-1kghejwzem.pdf

Sara E. Volz

Papers

Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

A Split Cas9 Architecture for Inducible Genome Editing and Transcription Modulation

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System

Multidimensional chemical control of CRISPR–Cas9

Multidimensional chemical control of CRISPR–Cas9