CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants

Abstract

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.

From Traditional Breeding to Genome Editing for Boosting Productivity of the Ancient Grain Tef [Eragrostis tef (Zucc.) Trotter]

“…SSNs such as CRISPR and CRISPR-associated protein 9 (CRISPR-Cas9) [ 117 , 118 , 119 , 120 ], transcriptional activator-like effector nuclease (TALEN) [ 121 , 122 , 123 ] and zinc finger nuclease (ZFN) [ 124 , 125 ] have been implicated in rapid genome editing in recent years. In addition to these, plant scientists use other techniques such as base editing, prime editing [ 126 ] and CRSIPR-Cpf1 [ 127 ]. Recently, CRISPR-Cpf1 has successfully used the prime genome editing in wheat Lin, et al [ 128 ] and rice Lin, et al [ 128 ], Li, et al [ 129 ] genomes.…”

Recent Advances in Genome Editing Tools in Medical Mycology Research

“…Prime editing, which can be used in biological systems, first searches for the points in the gene that need to be modified, then impose the editing effects, without breaking into double stranded DNA at the target points, through base deletion, insertion, and substitutions. The most notable advantage of this method to CRISPR is that it has less off-target effects and is more precise [ 115 , 116 , 117 ]. This technique is still in its infancy and has not yet been used in pathogenic fungi.…”

The Application of CRISPR/Cas9 Technology in the Management of Genetic and Nongenetic Plant Traits

“…Base editing is widely used in developing disease-resistant crops. Notwithstanding the previously described limitations above, base editing systems have the potential to simplify crop bioproduction or improvement, through high precision and efficiency [36]. Base editing could play a dynamic role in the introduction of gene mutations and directed protein evolution.…”

“…Base editing could play a dynamic role in the introduction of gene mutations and directed protein evolution. Cytosine base editing lacking UGI has the capacity to make diverse mutations, other than C-to-T. is ability has already been used in CRISPR-X and targeted AID-mediated mutagenesis-(TAM-) based studies to identify known and novel mutations in mammalian cells 2International Journal of Agronomy in cancer therapeutics targets PSMB5 and BCR-ABL, respectively [36]. Although there is an ever-increasing interest in BE and PE editing tools, there remain challenges that require further research, one major challenge being constrained target sites due to the PAM specificity of Cas proteins [31,33].…”