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 Acidominococcus 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.

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous RNAse 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 SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. We describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We use these engineered Cas9 activation complexes to investigate sgRNA targeting rules for effective transcriptional activation, demonstrate multiplexed activation of 10 genes simultaneously, and upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesize a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes which, upon activation, confer resistance to a BRAF inhibitor. Expected and potentially novel resistance genes are enriched in the top hits and are validated using individual sgRNA as well as cDNA overexpression. The signature of our top screening hits is significantly correlated with gene expression data from clinical melanoma samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.