Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

Rose, John C.; Popp, Nicholas; Richardson, Christopher D.; Stephany, Jason J.; Mathieu, Julie; Wei, Cindy T.; Corn, Jacob E.; Maly, Dustin J.; Fowler, Douglas M.

doi:10.1101/597849

Cited by 11 publications

(14 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Acr proteins offer an additional control point for implementing, for example spatio-temporal regulation in this type of circuits. Owing to its simplicity and its strong activity, Acr can result advantageous for circuit engineering in plants as compared to other dCas-offswitch methods such as those that rely on degron fusions to dCas (Kleinjan et al, 2017), or on catalytically inactivating gRNAs (Rose et al, 2020). One major issue when implementing inducible genetic circuits is transcriptional read-through or 'leaky' expression.…”

Section: Discussionmentioning

confidence: 99%

Strong and tunable anti‐CRISPR/Cas activities in plants

Calvache

Vázquez‐Vilar

Selma

et al. 2021

Plant Biotechnology Journal

View full text Add to dashboard Cite

CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.

show abstract

Section: Discussionmentioning

confidence: 99%

Strong and tunable anti‐CRISPR/Cas activities in plants

Calvache

Vázquez‐Vilar

Selma

et al. 2021

Plant Biotechnology Journal

View full text Add to dashboard Cite

show abstract

“…Combining the gRNA and Cas9 with a paired nickase significantly decreased the off-target effects in mammals [ 79 ]. Dead RNA off-target suppression (dOTS) is the latest developed strategy in which dead truncated gRNA that direct Cas9 binding, but suppress cleavage, has resulted in decreased off-target effects and increase on-target activity 40-fold [ 80 ].…”

Section: Mitigation Of Off-target Effects: Guide Rna (Grna) Modifimentioning

confidence: 99%

Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Naeem

Majeed

Hoque

et al. 2020

Cells

326

179

View full text Add to dashboard Cite

Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Numerous types of approaches have been developed to reduce off-target effects. Here, we review several latest approaches to reduce the off-target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs. This review article provides extensive information to cautiously interpret off-target effects to assist the basic and clinical applications in biomedicine.

show abstract

“…In summary, CRISPR GUARD has exciting potential for improving the specificity and safety of genome editing. During review, a similar technology report was published with complementary and supportive data 33 .…”

Section: Discussionmentioning

confidence: 99%

CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs

et al. 2020

View full text Add to dashboard Cite

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Gu ide RNA A ssisted R eduction of D amage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.

show abstract

Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

Cited by 11 publications

References 52 publications

Strong and tunable anti‐CRISPR/Cas activities in plants

Strong and tunable anti‐CRISPR/Cas activities in plants

Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs

Contact Info

Product

Resources

About