Genome editing in plants using CRISPR type I-D nuclease

Abstract

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

Genome editing in mammalian cells using the CRISPR type I-D nuclease

“…The results indicated that Cas3d protein exhibit weak ATPase activity (Figure 2A ). The results combined together with our previous study ( 37 ) suggest that both Cas10d and Cas3d cooperate for target DNA cleavage in the type I-D system.…”

“…PCC 6803 ( 32 ). We performed E. coli negative screening using a PAM library with the ccdB expression system, and identified that the type I-D from M. aeruginosa recognized only 5′-GTH-3′ (H = A, C or T) PAM in E. coli cells ( 37 ). We then examined whether GTT is active as the PAM in mammalian cells, and also analyzed the activity of GTC and GTA PAMs (Figure 4A , Supplementary Table S6 ; crRNAs listed in Supplementary Tables S2 and S6 ).…”

“…Therefore, the mode of interference through PAM recognition to target DNA degradation in the type I-D system must differ from that of other type I systems. Recently, we have successfully applied the type I-D system to plant genome editing ( 37 ), but this system has not yet been applied to mammalian genome editing.…”

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

“…With regard to the former, our detection method of biolistic delivery in pollen and pollinated pistils would contribute to increasing the efficiency at various reproductive steps. In the latter regard, a number of approaches aimed at enhancing the efficiency of genome editing have been reported, including the modification of Cas9 (Ling et al 2020; Osakabe et al 2020), design of guide RNA (Moon et al 2019), the use of RNPs (Liang et al 2018; Svitashev et al 2016), and the use of small chemical compounds (Yu et al 2015). Various selection methods have also been reported, including those based on antibiotic resistance (Chesnokov and Manteuffel 2000) and herbicide resistance, by targeting endogenous genes (Han and Kim 2019).…”

Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

“…As an alternative strategy, several Type I CRISPR-Cas3 systems belonging to Class 1 have been exploited to work as "built-in" genome editing tools in their native hosts (Zheng et al 2020;Xu et al 2021), including Type I-A of Sulfolobus islandicus (Li et al 2016), Type I-B of Haloarcula hispanica (Cheng et al 2017) and Clostridium species (Pyne et al 2016;Zhang et al 2018), Type I-C of Pectobacterium aeruginosa (Csorgo et al 2020), Type I-E of Streptococcus thermophilus (Canez et al 2019) and Lactobacillus crispatus (Hidalgo-Cantabrana et al 2019), and Type I-F of Pectobacterium species (Vercoe et al 2013;Xu et al 2019), and Zymomonas mobilis (Zheng et al 2019), where the processive Cas3 nuclease-helicase was used to generate chromosomal injuries. Recent studies have also employed Type I-D and I-E systems for DNA cleavage in plants (Osakabe et al 2020) and human cells (Cameron et al 2019;Dolan et al 2019;Morisaka et al 2019), respectively, and Type I-E and I-F systems for gene expression modulation in human cells (Pickar-Oliver et al 2019;Chen et al 2020), further broadening the applicability of CRISPR-Cas3-based technologies. These accomplishments have paved a new possibility to develop advanced CRISPR-nCas3 toolkits based on endogenous Type I systems.…”