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1. High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR /Cas9

   https://doi.org/10.1111/tpj.13446  

   Gil‐Humanes, Javier ; Wang, Yanpeng ; Liang, Zhen ; Shan, Qiwei ; Ozuna, Carmen V. ; Sánchez‐León, Susana ; Baltes, Nicholas J. ; Starker, Colby ; Barro, Francisco ; Gao, Caixia ; et al ( February 2017 , The Plant Journal)

   The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved usingAgrobacteriumT‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum(tobacco) andSolanum lycopersicum(tomato), greater than 10‐fold enhancements inGTfrequencies have been achieved usingDNAvirus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR/Cas9 nucleases and repair templates achievedGTat an endogenousubiquitinlocus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGTfrequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDVreplicons, multiplexedGTwithin the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGTusingWDV‐basedDNAreplicons will make it possible to edit complex cereal genomes without the need to integrateGTreagents into the genome.

    

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2. Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

   https://doi.org/10.1002/pld3.168  

   Mallett, Darren R. ; Chang, Mingqin ; Cheng, Xiaohang ; Bezanilla, Magdalena ( September 2019 , Plant Direct)

   CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping.

    

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3. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes

   https://doi.org/10.1126/science.aav4294  

   Harrington, Lucas B. ; Burstein, David ; Chen, Janice S. ; Paez-Espino, David ; Ma, Enbo ; Witte, Isaac P. ; Cofsky, Joshua C. ; Kyrpides, Nikos C. ; Banfield, Jillian F. ; Doudna, Jennifer A. ( October 2018 , Science)

   CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

    

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4. Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

   https://doi.org/10.3389/fmolb.2022.1072733  

   Wang, Jimin ; Arantes, Pablo R. ; Ahsan, Mohd ; Sinha, Souvik ; Kyro, Gregory W. ; Maschietto, Federica ; Allen, Brandon ; Skeens, Erin ; Lisi, George P. ; Batista, Victor S. ; et al ( January 2023 , Frontiers in Molecular Biosciences)

   The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences. 

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5. Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

   Wang, Jimin ; Arantes, Pablo R ; Ahsan, Mohd ; Sinha, Souvik ; Kyro, Gregory W ; Maschietto, Federica ; Allen, Brandon ; Skeens, Erin ; Lisi, George P. ; Batista, Victor S ; et al ( January 2023 , Frontiers in molecular biosciences)

   The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences. 

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