Manuscript Introduction - Plain Text

Writing the manuscript introduction
Adapted from MIT Biological Engineering Communication Lab: https://bit.ly/3tVXhtx.
General background.
Almost all archaea and many bacteria achieve adaptive immunity through a diverse set of
CRISPR-Cas (Clustered Regularly-Interspaced Short Palindromic Repeats and CRISPR-
ASsociated proteins) systems, each of which consists of a combination of Cas effector
proteins and CRISPR RNAs (crRNAs) (Makarova et al., 2011; Makarova et al., 2015).
Specific background (with all acronyms defined).

The defense activity of the CRISPR-Cas systems includes three stages: (i) adaptation, when a
complex of Cas proteins excises a segment of the target DNA (known as a protospacer) and
inserts it into the CRISPR array (where this sequence becomes a spacer); (ii) expression and
processing of the precursor CRISPR (pre-cr) RNA resulting in the formation of mature
crRNAs; and (iii) interference, when the effector module – either another Cas protein
complex or a single large protein – is guided by a crRNA to recognize and cleave target DNA
(or in some cases, RNA) (Horvath and Barrangou, 2010; Sorek et al., 2013; Barrangou and
Marraffini, 2014). The adaptation stage is mediated by the complex of the Cas1 and Cas2
proteins, which are shared by all known CRISPR-Cas systems, and sometimes involves
additional Cas proteins. Diversity is observed at the level of processing of the pre-crRNA to
mature crRNA guides, proceeding via either a Cas6-related ribonuclease or a housekeeping
RNaseIII that specifically cleaves double stranded RNA hybrids of pre-crRNA and tracrRNA.
Moreover, the effector modules differ substantially among the CRISPR-Cas systems
(Makarova et al., 2011; Makarova et al., 2015; Charpentier et al., 2015). In the latest
classification, the diverse CRISPR-Cas systems are divided into two classes according to the
configuration of their effector modules: Class 1 CRISPR systems utilize several Cas proteins
and the crRNA to form an effector complex, whereas Class 2 CRISPR systems employ a large
single-component Cas protein in conjunction with crRNAs to mediate interference (Makarova
et al., 2015). Multiple Class 1 CRISPR-Cas systems, which include the type I and type III
systems, have been identified and functionally characterized in detail, revealing the complex
architecture and dynamics of the effector complexes (Brouns et al., 2008; Marraffini and
Sontheimer, 2008; Hale et al., 2009; Sinkunas et al., 2013; Jackson et al., 2014; Mulepati et
al., 2014). Several Class 2 CRISPR-Cas systems have also been identified and experimentally
characterized, but they are all type II and employ homologous RNA-guided endonucleases of
the Cas9 family as effectors (Barrangou et al., 2007; Garneau et al., 2010; Deltcheva et al.,
2011; Sapranauskas et al., 2011; Jinek et al., 2012; Gasiunas et al., 2012).
Knowledge gap.
A second, putative Class 2 CRISPR system, tentatively assigned to type V, has been recently
identified in several bacterial genomes (Schunder et al., 2013; Vestergaard et al.,
2014; Makarova et al., 2015). The putative type V CRISPR-Cas systems contain a large,
~1,300 amino acid protein called Cpf1 (CRISPR from Prevotella and Francisella 1). It
remains unknown, however, if Cpf1-containing CRISPR loci indeed represent functional
CRISPR systems.
Why reader should care.

Given the broad applications of Cas9 as a genome engineering tool (Hsu et al., 2014; Jiang
and Marraffini, 2015), we sought to explore the function of Cpf1-based putative CRISPR
systems.
Statement of findings.
We found that the PAM for FnCpf1 is located upstream of the 5′ end of the displaced strand
of the protospacer and has the sequence 5′-TTN. […]
Here we show, with preview of results.

Here we show that Cpf1-containing CRISPR-Cas loci of Francisella tularensis subsp.
novicida U112encode functional defense systems capable of mediating plasmid interference
in bacterial cells guided by the CRISPR spacers. Unlike Cas9 systems, Cpf1-containing
CRISPR systems have three features: First, Cpf1-associated CRISPR arrays are processed
into mature crRNAs without the requirement of an additional trans-activating crRNA
(tracrRNA) (Deltcheva et al., 2011; Chylinski et al., 2013). Second, Cpf1-crRNA complexes
efficiently cleave target DNA proceeded by a short T-rich protospacer adjacent motif (PAM),
in contrast to the G-rich PAM following the target DNA for Cas9 systems. Third, Cpf1
introduces a staggered DNA double stranded break with a 4 or 5-nt 5′ overhang.
To explore the suitability of Cpf1 for genome editing applications, we characterized the RNA-
guided DNA targeting requirements for 16 Cpf1-family proteins from diverse bacteria, we
identify two Cpf1 enzymes, from Acidaminococcus sp. BV3L6 and Lachnospiraceae
bacterium ND2006, that are capable of mediating robust genome editing in human cells.
Why reader should care.

Collectively, these results establish a Class 2 CRISPR-Cas system that includes an effective
single RNA-guided endonuclease with distinct properties that has the potential to
substantially advance our ability to manipulate eukaryotic genomes.
Zetsche et al., Cell (2015), doi: 10.1016/j.cell.2015.09.038.

All rights reserved by Cell. Reproduced here for educational purposes only.
by @OpenAcademics

Manuscript Introduction - Plain Text

Uploaded by

Copyright:

Available Formats

Manuscript Introduction - Plain Text

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Manuscript Introduction - Plain Text

Uploaded by

Copyright:

Available Formats

Writing the manuscript introduction

Adapted from MIT Biological Engineering Communication Lab: https://bit.ly/3tVXhtx.

Specific background (with all acronyms defined).

Why reader should care.

Here we show, with preview of results.

Why reader should care.

Zetsche et al., Cell (2015), doi: 10.1016/j.cell.2015.09.038.

You might also like