Current and Prospective Applications of CRISPR-Cas12a in Pluricellular Organisms

Khan, Shaheen N.; Sallard, Erwan

doi:10.1007/s12033-022-00538-5

Cited by 8 publications

(16 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This might be explained by the fact that members of Cas12 and TnpB differ greatly in sequence similarity and length (33,34). The Cas12a group, which is currently actively studied and used in biotechnology applications (35), did not have thermostable (≥65 • C) sequences (Figure 6) as predicted by our method.…”

Section: Thermostability Of Class II Effector Proteinsmentioning

confidence: 76%

TemStaPro: protein thermostability prediction using sequence representations from protein language models

Pudžiuvelytė

Olechnovič

Godliauskaite³

et al. 2023

Preprint

View full text Add to dashboard Cite

Reliable prediction of protein thermostability from its sequence is valuable for both academic and industrial research. This prediction problem can be tackled using machine learning and by taking advantage of the recent blossoming of deep learning methods for sequence analysis. We propose applying the principle of transfer learning to predict protein thermostability using embeddings generated by protein language models (pLMs) from an input protein sequence. We used large pLMs that were pre-trained on hundreds of millions of known sequences. The embeddings from such models allowed us to efficiently train and validate a high-performing prediction method using over 2 million sequences that we collected from organisms with annotated growth temperatures. Our method, TemStaPro (Temperatures of Stability for Proteins), was used to predict thermostability of CRISPR-Cas Class II effector proteins (C2EPs). Predictions indicated sharp differences among groups of C2EPs in terms of thermostability and were largely in tune with previously published and our newly obtained experimental data. TemStaPro software is freely available from https://github.com/ievapudz/TemStaPro.

show abstract

Section: Thermostability Of Class II Effector Proteinsmentioning

confidence: 76%

TemStaPro: protein thermostability prediction using sequence representations from protein language models

Pudžiuvelytė

Olechnovič

Godliauskaite³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…The editing efficiency of Cas12a with a single gRNA was comparable to the efficiency of Cas9 with two to three gRNAs (21). The differences might result from i) the efficiency bias of Cas12a in penetrating the eggshell, ii) differences in the cleavage profiles of the nucleases (Cas9, blunt-ended; Cas12a, staggered ends) (30), iii) differences in the efficiency of NHEJ, which relies on the gRNA sequence and current cell-cycle state of the cells during treatment (103-105), and/or iv) combinations of these factors.…”

Section: Discussionmentioning

confidence: 99%

“…Correspondingly, increased transcript levels of HDR-associated genes during the parasitic life cycle was noted for the larval stages, including the sporocyst, which exhibit high rates of cell proliferation (Table S6) (79,120). By contrast, cells and tissue that are terminally differentiated without further need for proliferation, such as S4 vitelline cells, are likely to use NHEJ (105,118) for DNA repair (103,108).…”

Section: Discussionmentioning

confidence: 99%

ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT ASCHISTOSOMA MANSONIGENOME SAFE HARBOR

Moescheid,

Wisitphongpun,

Mann

et al. 2023

Preprint

View full text Add to dashboard Cite

Recently, we reported programmed Cas9 mediated insertion of a reporter gene into a gene safe harbor site, GSH1, ofSchistosoma mansonivia homology-directed repair (HDR) using overlapping guide RNAs. Here, we report efficient and precise CRISPR/Cas12a-mediated homology-directed insertion of a 5’ C6-PEG10-modified double-stranded transgene bearing microhomology arms, 50 nt length at GSH1. At the outset, we undertook bioinformatic and computational analysis following by experimental verification of the regulatory activity of schistosome ubiquitin (SmUbi) promoter and terminator, aiming to drive strong reporter gene expression. Green fluorescent protein activity driven by this endogenous promoter followed electroporation-mediated transfection of schistosome eggs. HDR induced by CRISPR/Cas12a delivered more efficient transgene integration compared to CRISPR/Cas9, with precise integration of the transgene at Cas12a cleavage sites, which releases overhanding DNA strands at 18-24 nt during programmed cleavage. The transfection design for CRISPR/Cas-mediated genome editing facilitated precise knock-in, specifically chromosomal integration of the SmUbi-EGFP-SmUbi reporter-gene with microhomology arms into GSH1. In this non-model system, the 5’-C6-PEG10 modified-DNA template enhanced knockin efficiency of Cas9 and Cas12a. This approach advances schistosome transgenesis and may be functional also in related parasitic and non-parasitic helminths, which hitherto lack functional genomics and transfection methods.GRAPHICAL ABSTRACT

show abstract

“…In addition to the widely used Cas9, Cas12a is often used for multiple gene editing in plant genomes [ 181 ]. However, the CRISPR/Cas12a system shows different editing efficiencies at genomic loci and is significantly affected by CRISPR RNA (crRNA) [ 182 , 183 , 184 ].…”

Section: Strategies and Methods For Optimizing Crispr/cas9 Gene-editi...mentioning

confidence: 99%

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Zhou

Luan

Liu

et al. 2023

Plants

View full text Add to dashboard Cite

Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

show abstract

Current and Prospective Applications of CRISPR-Cas12a in Pluricellular Organisms

Cited by 8 publications

References 71 publications

TemStaPro: protein thermostability prediction using sequence representations from protein language models

TemStaPro: protein thermostability prediction using sequence representations from protein language models

ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT ASCHISTOSOMA MANSONIGENOME SAFE HARBOR

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Contact Info

Product

Resources

About