Andrew V. Anzalone, Peyton B. Randolph, Jessie R. Davis and David R. Liu
This paper introduced prime editing, a versatile genome-editing method that uses a Cas9 nickase fused to a reverse transcriptase guided by a prime editing guide RNA (pegRNA) to write new genetic information directly into a target site. Without requiring double-strand breaks or donor DNA, prime editing can install targeted insertions, deletions, and all 12 types of point mutations. The authors demonstrated correction of disease-relevant mutations in human cells with broad targeting flexibility and relatively low off-target activity.
Nicole M. Gaudelli, Alexis C. Komor, Holly A. Rees and David R. Liu
This work developed adenine base editors (ABEs) by evolving a transfer RNA adenosine deaminase to act on DNA, enabling direct conversion of A-T base pairs to G-C in genomic DNA without double-strand breaks. Because no natural DNA adenosine deaminase was available, the authors used directed evolution to create the enzyme, then fused it to Cas9 nickase. ABEs corrected target adenines efficiently and with high product purity and low indel formation in human cells.
Omar O. Abudayyeh, Jonathan S. Gootenberg, Patrick Essletzbichler and Feng Zhang
This study characterized the class 2 type VI CRISPR effector Cas13a (formerly C2c2) as a programmable RNA-targeting tool in mammalian and plant cells. A catalytically inactive Cas13a (dCas13a) was used for RNA binding while active Cas13a enabled efficient, specific knockdown of endogenous transcripts. The authors showed RNA knockdown comparable to or more specific than RNA interference and demonstrated applications such as transcript tracking and splicing modulation.
Jonathan S. Gootenberg, Omar O. Abudayyeh, Jeong Wook Lee and Feng Zhang
This paper introduced SHERLOCK, a CRISPR-based nucleic acid detection platform built on the collateral RNase activity of Cas13a (C2c2). Upon recognizing a target sequence, Cas13a indiscriminately cleaves nearby reporter RNAs; combined with isothermal amplification, this yields highly sensitive, specific detection. The authors demonstrated attomolar sensitivity and single-base discrimination, with applications in detecting viruses (Zika, dengue), bacteria, and human genotypes.
Alexis C. Komor, Yongjoo B. Kim, Michael S. Packer, John A. Zuris and David R. Liu
This paper introduced cytosine base editing (CBE), a strategy that fuses a catalytically impaired Cas9 to a cytidine deaminase to directly convert C-G base pairs to T-A in genomic DNA without inducing double-strand breaks or requiring a donor template. The authors showed that within a programmable target window the deaminase converts cytosine to uracil, which is then read as thymine, and that inhibiting base-excision repair markedly improves editing efficiency. The approach achieved precise single-base correction in human and other mammalian cells.
Bernd Zetsche, Jonathan S. Gootenberg and Feng Zhang
This paper characterizes Cpf1 (later named Cas12a) as a single-RNA-guided DNA endonuclease of a class 2 CRISPR-Cas system, expanding the genome-editing toolbox beyond Cas9. Cpf1 requires only a single crRNA (no tracrRNA), recognizes a T-rich PAM, and produces staggered cuts with overhangs. The authors demonstrate Cpf1-mediated genome editing in human cells.
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna and Emmanuelle Charpentier
This study demonstrated that the CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease whose target specificity is determined by a dual-RNA structure formed by a CRISPR RNA (crRNA) base-paired to a trans-activating crRNA (tracrRNA). The authors showed that Cas9 introduces site-specific double-strand breaks in target DNA, with its HNH domain cleaving the complementary strand and its RuvC-like domain cleaving the noncomplementary strand. Critically, they engineered the two guide RNAs into a single chimeric guide RNA that still directed sequence-specific cleavage, establishing the system as a programmable tool for genome editing.