Match!

CRISPR adenine and cytosine base editors with reduced RNA off-target activities

Published on May 9, 2019in bioRxiv
· DOI :10.1101/631721
Julian Grünewald2
Estimated H-index: 2
(Harvard University),
Ronghao Zhou2
Estimated H-index: 2
(Harvard University)
+ 4 AuthorsJ. Keith Joung62
Estimated H-index: 62
(Harvard University)
Abstract
Abstract CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively1-4. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies5. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing5. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
  • References (20)
  • Citations (3)
References20
Newest
#1Julian GrünewaldH-Index: 2
#2Ronghao Zhou (Harvard University)H-Index: 2
Last.J. Keith JoungH-Index: 62
view all 7 authors...
27 CitationsSource
#1Kendell Clement (Broad Institute)H-Index: 16
#2Holly A. Rees (Broad Institute)H-Index: 10
Last.Luca Pinello (Broad Institute)H-Index: 22
view all 11 authors...
18 CitationsSource
#1Holly A. Rees (Broad Institute)H-Index: 10
#2David R. Liu (Broad Institute)H-Index: 73
75 CitationsSource
#1Jason M. GehrkeH-Index: 3
Last.J. Keith JoungH-Index: 62
view all 8 authors...
59 CitationsSource
#1Xiao WangH-Index: 2
#2Jianan LiH-Index: 8
Last.Li YangH-Index: 33
view all 10 authors...
31 CitationsSource
#1Luke W. KoblanH-Index: 3
#2Jordan L. DomanH-Index: 3
Last.David R. LiuH-Index: 73
view all 9 authors...
67 CitationsSource
#1Nicole M. GaudelliH-Index: 6
#2Alexis C. KomorH-Index: 13
Last.David R. LiuH-Index: 73
view all 7 authors...
457 CitationsSource
#1Alexis C. Komor (Broad Institute)H-Index: 13
#2Kevin T. Zhao (Broad Institute)H-Index: 5
Last.David R. Liu (Broad Institute)H-Index: 73
view all 9 authors...
126 CitationsSource
#1Shraddha Sharma (Roswell Park Cancer Institute)H-Index: 6
#2Santosh K. Patnaik (Roswell Park Cancer Institute)H-Index: 14
Last.Bora E. Baysal (Roswell Park Cancer Institute)H-Index: 31
view all 4 authors...
21 CitationsSource
#1Keiji Nishida (Kobe University)H-Index: 24
#2Takayuki Arazoe (Kobe University)H-Index: 7
Last.Akihiko Kondo (Kobe University)H-Index: 70
view all 12 authors...
335 CitationsSource
Cited By3
Newest
#1Rina C. Sakata (UTokyo: University of Tokyo)
#2Soh Ishiguro (UTokyo: University of Tokyo)H-Index: 2
Last.Nozomu YachieH-Index: 17
view all 13 authors...
Source
#1Kai Hua (CAS: Chinese Academy of Sciences)H-Index: 5
#2Jinshan Zhang (CAS: Chinese Academy of Sciences)H-Index: 6
Last.Jian-Kang Zhu (CAS: Chinese Academy of Sciences)H-Index: 139
view all 7 authors...
Source
#1Changyang Zhou (CAS: Chinese Academy of Sciences)H-Index: 5
#2Yidi Sun (CAS: Chinese Academy of Sciences)H-Index: 8
Last.Hui Yang (CAS: Chinese Academy of Sciences)H-Index: 19
view all 14 authors...
9 CitationsSource