More Like this

1. Are there increases in CRISPR/Cas off-target effects in homologous recombination repair deficient Saccharomyces cerevisiae cells?

   Castillo, S; Santoro, I (January 2023, Southeastern biology)

   CRISPR/Cas technology is increasingly being used as a common methodology in many cancer biology studies due to the ease and convenience of the technique. Precise editing of genomic DNA has been achieved upon repair of CRISPR-induced DNA double-strand breaks (DSBs) by homologous recombination (HR). HR repairs DNA DSBs with high fidelity and therefore, deficiencies in HR result in genome instability. These deficiencies have been demonstrated in many cancers. RAD51-dependent HR is a very important pathway for repairing DSBs. Previous studies have shown that genome editing using CRISPR technology relies on the repair of site-specific DNA DSBs induced by the RNA-guided Cas9 endonuclease. Furthermore, previous studies have shown that the efficiency of CRISPR-mediated HR can be improved by the stimulation of HR promoting factors, such as the RAD51 recombinase. Despite the ease and efficient use the CRISPR/Cas technology for genome editing, one limitation is the potential occurrence of associated off-target effects. If CRISPR technology is planned to be used to target cancer cells with defective HR capabilities, will off-target mutations be likely to occur? In order to answer this question, a system was developed in Saccharomyces cerevisiae using green fluorescent protein (GFP) as a reporter to identify off-target CRISPR-induced DSBs. This study set out to test the number of off-target DSBs that could be introduced by CRISPR-induced genome editing in a RAD51-deficient HR model. We were curious whether loss of RAD51-dependent HR would increase the abundance of off-target CRISPR-induced DSBs in mutant yeast strains as compared to those with a functioning HR-dependent DNA repair pathway. Preliminary findings using this system will be presented. 

   more » « less
2. Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites

   https://doi.org/10.1038/s41556-022-00975-z  

   Zou, Roger S.; Marin-Gonzalez, Alberto; Liu, Yang; Liu, Hans B.; Shen, Leo; Dveirin, Rachel K.; Luo, Jay X. J.; Kalhor, Reza; Ha, Taekjip (September 2022, Nature Cell Biology)

   Abstract Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci. 

   more » « less
3. 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)

   Abstract 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. 

   more » « less
4. Epigenetic features drastically impact CRISPR–Cas9 efficacy in plants

   https://doi.org/10.1093/plphys/kiac285  

   Weiss, Trevor; Crisp, Peter A.; Rai, Krishan M.; Song, Meredith; Springer, Nathan M.; Zhang, Feng (June 2022, Plant Physiology)

   Abstract CRISPR–Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR–Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR–Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR–Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR–Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes. 

   more » « less
5. Reversible RNA acylation for control of CRISPR–Cas9 gene editing

   https://doi.org/10.1039/C9SC03639C  

   Habibian, Maryam; McKinlay, Colin; Blake, Timothy R.; Kietrys, Anna M.; Waymouth, Robert M.; Wender, Paul A.; Kool, Eric T. (January 2020, Chemical Science)

   null (Ed.)

   We report the development of post-transcriptional chemical methods that enable control over CRISPR–Cas9 gene editing activity both in in vitro assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N 3 ) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated (“cloaked”) sgRNA was completely inactive in DNA cleavage with Cas9 in vitro , and activity was quantitatively restored after phosphine treatment. Delivery of cloaked sgRNA and Cas9 mRNA into HeLa cells was enabled by the use of charge-altering releasable transporters (CARTs), which outperformed commercial transfection reagents in transfecting sgRNA co-complexed with Cas9 encoding functional mRNA. Genomic DNA cleavage in the cells by CRISPR–Cas9 was efficiently restored after treatment with phosphine to remove the blocking acyl groups. Our results highlight the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function. 

   more » « less