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In this randomized, controlled trial, the number of angioedema attacks per month was approximately 75% lower among adults with hereditary angioedema who received a CRISPR-Cas9–based therapy than among those who received placebo.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins 1 . Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases 2 , 3 . TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR–Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA–target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR–Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR–Cas12 effectors. Cryo-electron microscopy analysis of the Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA provides insights into the mechanism of TnpB function and the evolution of CRISPR–Cas12 effectors.

The CRISPR-associated protein Cpf1 from Francisella novicida is a novel enzyme with specific, dual-endoribonuclease–endonuclease activities in precursor crRNA processing and crRNA-programmable cleavage of target DNA. Cpf1 enzyme in CRISPR immunity The bacterial immune system, CRISPR, utilizes a small RNA guide, or crRNA, to target a nucleolytic CRISPR complex to DNA with a complementary sequence. This process has been widely exploited for various types of genome engineering. Previously described CRISPR systems utilize one nuclease, such as Cas6, to generate the mature crRNA, and a second, such as Cas9, to cleave the target DNA. Two studies illustrate a different approach that involves the Cpf1 protein. Emmanuelle Charpentier and colleagues report that type V-A Cpf1 protein from Francisella novicida functions as a minimalistic CRISPR system. It is a dual-nuclease enzyme that can perform both the pre-crRNA processing and DNA cleavage activities, having distinct active domains for the two substrates. Zhiwei Huang and colleagues solve the crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to crRNA, showing how binding induces conformational changes in the nuclease. CRISPR–Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA 1 . The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) 2 , 3 , 4 , 5 and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA 6 , 7 , 8 , 9 , 10 , 11 , 12 . In type II systems, RNase III cleaves pre-crRNA base-paired with trans -activating crRNA (tracrRNA) in the presence of Cas9 (refs 13 , 14 ). The mature tracrRNA–crRNA duplex then guides Cas9 to cleave target DNA 15 . Here, we demonstrate a novel mechanism in CRISPR–Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5′-YTN-3′ protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5′ overhang 16 . The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR–Cas systems so far described.

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9–based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T . thermophilus ) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis ( S. epidermidis ) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes. Type III CRISPR-Cas systems are able to target transcriptionally active DNA sequences in phages and plasmids. Here, the authors reveal the mechanism of the target nucleic acid preference of Type III-A CRISPR-Cas complexes at the transcription bubble by a combination of structural and biochemical approaches.

In the presence of a short DNA oligonucleotide containing a protospacer adjacent motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification. A technology for RNA recognition The bacterial CRISPR immune defence system, and its effector Cas9 in particular, have recently been exploited for sequence-specific genome editing in eukaryotic cells. Cas9 binds a guide RNA and in the presence of a DNA motif known as protospacer adjacent motif (PAM), is able to cleave the target DNA. New work by Jennifer Doudna and colleagues reveals the unexpected result that in the presence of a DNA oligomer containing PAM, a guide RNA-programmed Cas9 is able to cleave single-stranded RNA as well. They show that this system can also be used to isolate specific endogenous RNA transcripts, without any tag or modification, from a cell lysate. Thus, the system can be programmed to either bind or cut desired RNA targets, depending on the PAM used. This work and points the way towards possible new technologies for programmable RNA recognition. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage 1 , 2 , 3 , 4 , 5 . In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA 4 , 5 , 6 , 7 . Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms 8 , but it has been thought to be incapable of targeting RNA 5 . Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage 7 . Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

The crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to CRISPR RNA is presented, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing. Cpf1 enzyme in CRISPR immunity The bacterial immune system, CRISPR, utilizes a small RNA guide, or crRNA, to target a nucleolytic CRISPR complex to DNA with a complementary sequence. This process has been widely exploited for various types of genome engineering. Previously described CRISPR systems utilize one nuclease, such as Cas6, to generate the mature crRNA, and a second, such as Cas9, to cleave the target DNA. Two studies illustrate a different approach that involves the Cpf1 protein. Emmanuelle Charpentier and colleagues report that type V-A Cpf1 protein from Francisella novicida functions as a minimalistic CRISPR system. It is a dual-nuclease enzyme that can perform both the pre-crRNA processing and DNA cleavage activities, having distinct active domains for the two substrates. Zhiwei Huang and colleagues solve the crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to crRNA, showing how binding induces conformational changes in the nuclease. The CRISPR–Cas systems, as exemplified by CRISPR–Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection 1 , 2 , 3 , 4 , 5 , 6 , 7 . The CRISPR–Cpf1 system, a new class 2 CRISPR–Cas system, mediates robust DNA interference in human cells 1 , 8 , 9 , 10 . Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H 2 O) 6 ) 2+ ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.

Guide RNAs with shorter regions of complementarity to target sites reduce off-target effects. Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools 1 , 2 , 3 . CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target 4 , 5 , 6 . Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.

Programmable sequence-specific genome editing agents such as CRISPR-Cas9 have greatly advanced our ability to manipulate the human genome. Although canonical forms of genome-editing agents and programmable transcriptional regulators are constitutively active, precise temporal and spatial control over genome editing and transcriptional regulation activities would enable the more selective and potentially safer use of these powerful technologies. Here, by incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, we developed a set of aptazyme-embedded guide RNAs that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells. CRISPR-Cas9 is a powerful technique for manipulating the human genome, however temporal and spatial control of activity would facilitate safer, more selective use. Here the authors incorporate aptazymes into guide RNAs to allow small molecule activation of CRISPR-Cas9.

Engineered Sp Cas9s and As Cas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq—nuclease digestion and deep sequencing—a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five Sp Cas9 variants and As Cas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, As Cas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases. The enzymatic properties of RNA-guided nucleases are revealed through massively parallel analysis.