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\"The bestselling author of Leonardo da Vinci and Steve Jobs returns with a gripping account of how the pioneering scientist Jennifer Doudna, along with her colleagues and rivals, launched a revolution that will allow us to cure diseases, fend off viruses, and enhance our children\"-- Provided by publisher.

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.

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.

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.

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR–Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage 1 . Although a few eukaryotic RNA-guided systems have been studied, including RNA interference 2 and ribosomal RNA modification 3 , it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported 4 , 5 . The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity 4 , 6 . TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins 4 , 7 , raising the possibility that eukaryotes are also equipped with CRISPR–Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life. Fanzor is shown to be an RNA-guided DNA endonuclease, demonstrating that such endonucleases are found in all domains of life and indicating a potential new tool for genome engineering applications.

Microphthalmia is a congenital ocular disorder resulting from abnormal morphogenesis during early eye development. It affects approximately 17 in every 100,000 live births and accounts for up to 11% of childhood blindness worldwide. Unfortunately, there’s currently no treatment available. Clinical data shows that gene mutations are associated with the development of microphthalmia. Here, we particularly focus on FNBP4 (formin binding protein 4). Whole-genome sequencing identified a homozygous FNBP4 mutation in microphthalmia patients. Despite its identification, research on FNBP4 and its role in eye development remains limited. Preliminary study in our lab revealed that CRISPR knockout of FNBP4 results in microphthalmia in zebrafish embryos. Additionally, FNBP4 knockout leads to increased apoptosis and an expansion of phospho-Smad1/5/8 staining in the embryonic eye implicating alterations in cellular dynamics and BMP signalling as a potential mechanism underlying microphthalmia conditions. Therefore, this project aims to explore the molecular mechanism of FNBP4 in eye development by identifying binding partners and investigating FNBP4 regulation of the BMP-SMAD signalling pathway in retinal cell-lines and iPSC-derived organoids. Western blot analysis revealed that FNBP4 knockdown in RPE-1 cells increases phosphorylated Smad (p-Smad1/5/8) levels in response to BMP4 stimulation, suggesting a conserved role for FNBP4 in inhibiting or attenuating BMP signalling. To further investigate the cellular pathways regulated by FNBP4, we will employ RNA-seq and Co-IP in RPE cell lines and generate wild-type and FNBP4 CRISPR-knockout hiPSC-derived retinal organoids to examine its role in ocular development.

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. CRISPR-Cas12a is a promising system for targeting AT-rich regions of the genome. Here the authors identify Cas12a orthologs with expanded targeting scope and develop a highly multiplexable editing system in rice.

The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences. One of the key limitations of CRISPR-Cas-based genome editing techniques is the PAM dependency. Here, the authors review ongoing efforts towards realizing PAM-free nucleases, address potential consequences of eliminating PAM recognition, and propose an alternative nuclease repertoire covering all possible PAM sequences.