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Overcoming the AAV Size Limitation for CRISPR Delivery

Posted by Mary Gearing on Sep 16, 2020 9:00:00 AM

Originally published Jul 14, 2015 and last updated Sep 16, 2020 by Beth Kenkel. 

CRISPR genome editing has quickly become a popular system for in vitro and germline genome editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb). While this approach has been proven feasible, it leaves little room for additional regulatory elements. Feng Zhang's group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating purification and co-infection of two AAVs.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

CRISPR 101: Multiplex Expression of gRNAs

Posted by Mary Gearing on Sep 10, 2020 7:45:00 AM

Originally published Jan 28, 2016 and last updated Sep 10, 2020 by Jennifer Tsang.

CRISPR makes it easy to target multiple loci - a concept called multiplexing. Since CRISPR is such a robust system, editing or labeling efficiency doesn’t usually change when you add multiple gRNAs on one plasmid. Sound good? Addgene has many tools to help you multiplex - we’ll use mammalian plasmids to introduce you to some of your potential options and cloning methods, but please scroll down for plasmids suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!

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Topics: CRISPR, CRISPR 101, CRISPR gRNAs

Optogenetics + CRISPR, Using Light to Control Genome Editing

Posted by Caroline LaManna on Sep 3, 2020 8:15:00 AM

Originally published Mar 8, 2016 and last updated Sept 3, 2020 by Nyla Naim.

Scientists around the world have been making major improvements to CRISPR technology since its initial applications for genome engineering in 2012.  Many of these advances have stemmed from the goal of reducing off-target Cas9 activity. The use of nickases, prime editing, anti-CRISPR proteins, and other techniques all aim to improve targeting specificity or reduce the duration of Cas9 activity. 

The field of optogenetics is renowned for enabling precise temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular activities using light. In 2015, scientists combined CRISPR and optogenetic techniques to develop a variety of photoactivatable CRISPR tools. These tools allow scientists to use light to externally control the location, timing, and reversibility of the genome editing process. Read on to learn about the various light-controlled CRISPR tools available to researchers - some readily found at Addgene.

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Topics: Optogenetics, CRISPR, Other CRISPR Tools

The PAM Requirement and Expanding CRISPR Beyond SpCas9

Posted by Joel McDade on Aug 20, 2020 10:30:00 AM

Originally published Nov 12, 2015 and last updated Aug 20, 2020.

Cas9 can be used to modify any desired genomic target provided that (1) the sequence is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage.

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Topics: CRISPR, Cas Proteins

CRISPR 101: Cytosine and Adenine Base Editors

Posted by Mary Gearing on Aug 6, 2020 10:30:00 AM

Originally published Aug 16, 2016 and last updated Aug 6, 2020.

When we talk about CRISPR applications, one negative often comes up: the low editing efficiency of homology-directed repair (HDR). Compared to non-homologous end joining, HDR occurs at a relatively low frequency, and in nondividing cells, this pathway is further downregulated. Rather than try to improve HDR, scientists have developed two classes of base editors: cytosine base editors (CBEs) and adenine base editors (ABEs). 

(There are also RNA base editors, but we’ll just be covering DNA base editors here. To learn more about RNA base editors head over to this blog post: CRISPR 101: RNA Editing with Cas13)

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Topics: CRISPR, Base Editing

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